Bakkeren Molecular Plant Pathology Lab

Publication Abstracts

 

Yadav et al. 2018. RNAi is a critical determinant of centromere evolution in closely related fungi. Proc. Natl. Acad. Sci. USA 115(12): 3108-3113. DOI: 10.1073/pnas.1713725115.

Abstract: The “centromere paradox” refers to rapidly evolving and highly diverse centromere DNA sequences even in closely related eukaryotes. However, factors contributing to this rapid divergence are largely unknown. Here, we identified large regional, LTR retrotransposon-rich centromeres in a group of human fungal pathogens belonging to the Cryptococcus species complex. We provide evidence that loss-of-functional RNAi machinery and possibly cytosine DNA methylation trigger instability of the genome by activation of centromeric retrotransposons presumably suppressed by RNAi. We propose that RNAi, together with cytosine DNA methylation, serves as a critical determinant that maintains repetitive transposon-rich centromere structures. This study explores the direct link between RNAi and centromere structure evolution.The centromere DNA locus on a eukaryotic chromosome facilitates faithful chromosome segregation. Despite performing such a conserved function, centromere DNA sequence as well as the organization of sequence elements is rapidly evolving in all forms of eukaryotes. The driving force that facilitates centromere evolution remains an enigma. Here, we studied the evolution of centromeres in closely related species in the fungal phylum of Basidiomycota. Using ChIP-seq analysis of conserved inner kinetochore proteins, we identified centromeres in three closely related Cryptococcus species: two of which are RNAi-proficient, while the other lost functional RNAi. We find that the centromeres in the RNAi-deficient species are significantly shorter than those of the two RNAi-proficient species. While centromeres are LTR retrotransposon-rich in all cases, the RNAi-deficient species lost all full-length retroelements from its centromeres. In addition, centromeres in RNAi-proficient species are associated with a significantly higher level of cytosine DNA modifications compared with those of RNAi-deficient species. Furthermore, when an RNAi-proficient Cryptococcus species and its RNAi-deficient mutants were passaged under similar conditions, the centromere length was found to be occasionally shortened in RNAi mutants. In silico analysis of predicted centromeres in a group of closely related Ustilago species, also belonging to the Basidiomycota, were found to have undergone a similar transition in the centromere length in an RNAi-dependent fashion. Based on the correlation found in two independent basidiomycetous species complexes, we present evidence suggesting that the loss of RNAi and cytosine DNA methylation triggered transposon attrition, which resulted in shortening of centromere length during evolution.

 

Panwar et al. 2018. Host-induced silencing of essential genes in Puccinia triticina through transgenic expression of RNAi sequences reduces severity of leaf rust infection in wheat. Plant Biotechnol. J. 16(5): 1013-1023. DOI:10.1111/pbi.12845.

Abstract: Leaf rust, caused by the pathogenic fungus Puccinia triticina (Pt), is one of the most serious biotic threats to sustainable wheat production worldwide. This obligate biotrophic pathogen is prevalent worldwide and is known for rapid adaptive evolution to overcome resistant wheat varieties. Novel disease control approaches are therefore required to minimize the yield losses caused by Pt. Having shown previously the potential of host-delivered RNA interference (HD-RNAi) in functional screening of Pt genes involved in pathogenesis, we here evaluated the use of this technology in transgenic wheat plants as a method to achieve protection against wheat leaf rust (WLR) infection. Stable expression of hairpin RNAi constructs with sequence homology to Pt MAP Kinase (PtMAPK1) or a cyclophilin (PtCYC1) encoding gene in susceptible wheat plants showed efficient silencing of the corresponding genes in the interacting fungus resulting in disease resistance throughout the T2 generation. Inhibition of Pt proliferation in transgenic lines by in planta-induced RNAi was associated with significant reduction in target fungal transcript abundance and reduced fungal biomass accumulation in highly resistant plants. Disease protection was correlated with the presence of siRNA molecules specific to targeted fungal genes in the transgenic lines harbouring the complementary HD-RNAi construct. This work demonstrates that generating transgenic wheat plants expressing RNAi-inducing transgenes to silence essential genes in rust fungi can provide effective disease resistance, thus opening an alternative way for developing rust-resistant crops. This article is protected by copyright. All rights reserved.

 

Coelho et al. 2017. 2017. Fungal Sex: The Basidiomycota. Microbiology Spectrum 5(3). DOI:10.1128/microbiolspec.FUNK-0046-2016.

Abstract: Fungi of the Basidiomycota, representing major pathogen lineages and mushroom-forming species, exhibit diverse means to achieve sexual reproduction, with particularly varied mechanisms to determine compatibilities of haploid mating partners. For species that require mating between distinct genotypes, discrimination is usually based on both the reciprocal exchange of diffusible mating pheromones, rather than sexes, and the interactions of homeodomain protein signals after cell fusion. Both compatibility factors must be heterozygous in the product of mating, and genetic linkage relationships of the mating pheromone/receptor and homeodomain genes largely determine the complex patterns of mating-type variation. Independent segregation of the two compatibility factors can create four haploid mating genotypes from meiosis, referred to as tetrapolarity. This condition is thought to be ancestral to the basidiomycetes. Alternatively, cosegregation by linkage of the two mating factors, or in some cases the absence of the pheromone-based discrimination, yields only two mating types from meiosis, referred to as bipolarity. Several species are now known to have large and highly rearranged chromosomal regions linked to mating-type genes. At the population level, polymorphism of the mating-type genes is an exceptional aspect of some basidiomycete fungi, where selection under outcrossing for rare, intercompatible allelic variants is thought to be responsible for numbers of mating types that may reach several thousand. Advances in genome sequencing and assembly are yielding new insights by comparative approaches among and within basidiomycete species, with the promise to resolve the evolutionary origins and dynamics of mating compatibility genetics in this major eukaryotic lineage.

 

Panwar and Bakkeren. 2017. Investigating gene function in cereal rust fungi by plant-mediated Virus-Induced Gene Silencing. Wheat rust diseases: Methods in Molecular Biology, Vol. 1659. S. Periyannan. New York, NY, Humana Press, Springer: pp. 115-124. DOI:10.1007/978-1-4939-7249-4_10

Abstract: Cereal rust fungi are destructive pathogens, threatening grain production worldwide. Targeted breeding for resistance utilizing host resistance genes has been effective. However, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to expand the range of available resistance genes. Whole genome sequencing, transcriptomic and proteomic studies followed by genome-wide computational and comparative analyses have identified large repertoire of genes in rust fungi among which are candidates predicted to code for pathogenicity and virulence factors. Some of these genes represent defence triggering avirulence effectors. However, functions of most genes still needs to be assessed to understand the biology of these obligate biotrophic pathogens. Since genetic manipulations such as gene deletion and genetic transformation are not yet feasible in rust fungi, performing functional gene studies is challenging. Recently, Host-induced gene silencing (HIGS) has emerged as a useful tool to characterize gene function in rust fungi while infecting and growing in host plants. We utilized Barley stripe mosaic virus-mediated virus induced gene silencing (BSMV-VIGS) to induce HIGS of candidate rust fungal genes in the wheat host to determine their role in plant–fungal interactions. Here, we describe the methods for using BSMV-VIGS in wheat for functional genomics study in cereal rust fungi.

 

Cuomo et al. 2017. Comparative analysis highlights variable genome content of wheat rusts and divergence of the mating loci. G3: Genes|Genomes|Genetics. 7(2): 361-376. DOI:10.1534/g3.116.032797

Abstract: Three members of the Puccinia genus, Puccinia triticina (Pt), P. striiformis f.sp. tritici (Pst), and P. graminis f.sp. tritici (Pgt), cause the most common and often most significant foliar diseases of wheat. While similar in biology and life cycle, each species is uniquely adapted and specialized. The genomes of Pt and Pst were sequenced and compared to that of Pgt to identify common and distinguishing gene content, to determine gene variation among wheat rust pathogens, other rust fungi, and basidiomycetes, and to identify genes of significance for infection. Pt had the largest genome of the three, estimated at 135 Mb with expansion due to mobile elements and repeats encompassing 50.9% of contig bases; in comparison, repeats occupy 31.5% for Pst and 36.5% for Pgt. We find all three genomes are highly heterozygous, with Pst [5.97 single nucleotide polymorphisms (SNPs)/kb] nearly twice the level detected in Pt (2.57 SNPs/kb) and that previously reported for Pgt. Of 1358 predicted effectors in Pt, 784 were found expressed across diverse life cycle stages including the sexual stage. Comparison to related fungi highlighted the expansion of gene families involved in transcriptional regulation and nucleotide binding, protein modification, and carbohydrate degradation enzymes. Two allelic homeodomain pairs, HD1 and HD2, were identified in each dikaryotic Puccinia species along with three pheromone receptor (STE3) mating-type genes, two of which are likely representing allelic specificities. The HD proteins were active in a heterologous Ustilago maydis mating assay and host-induced gene silencing (HIGS) of the HD and STE3 alleles reduced wheat host infection.

 

Panwar et al. 2016. RNA silencing approaches for identifying pathogenicity and virulence elements towards engineering crop resistance to plant pathogenic fungi. In: CABI Reviews. Perspectives in Agriculture, Veterinary Science, Nutrition and Natural Resources. 11(027). DOI:10.1079/PAVSNNR201611027

Abstract: Over the past several years, RNA interference (RNAi) has emerged as a powerful genetic tool that is being exploited not only in fundamental research for the assessment of gene functions, but also in various fields of applied research in medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content, but also for plant protection. In the study of plant–fungus interactions, a comprehensive understanding of pathogenic mechanisms based on information from genome sequences demands advanced genomics tools and infrastructure. In order to make use of the plethora of information yielded by the rapidly increasing fungal pathogen genome sequencing studies, there is a growing need to achieve gene function assessment amenable to high-throughput techniques. Targeted gene disruption is effective but may not be feasible in certain fungi because of lack of transformation protocols or their life styles. RNAi techniques offer an alternative but, in contrast to other biological systems, only few studies have reported the use of RNAi to explore gene function in plant pathogenic fungi. In this review, we summarize a variety of options using RNAi techniques that have become available to study gene function for high-throughput phenotypic screening of virulence and pathogenicity factors in plant fungi. The identification of such genes can be exploited for the protection of crops against fungal pathogens.

 

Cervantes-Chávez et al. 2016. Trehalose is required for stress resistance and virulence of the Basidiomycota plant pathogen Ustilago maydis. Microbiology. 162(6): 1009-1022. DOI:10.1099/mic.0.000287

Abstract: Trehalose is an important disaccharide that can be found in bacteria, fungi, invertebrates and plants. In some Ascomycota fungal plant pathogens, the role of trehalose was recently studied and shown to be important for conferring protection against several environmental stresses and for virulence. In most of the fungi studied, two enzymes are involved in the synthesis of trehalose: trehalose-6 phosphate synthase (Tps1) and trehalose-6 phosphate phosphatase (Tps2). To study the role of trehalose in virulence and stress response in the Basidiomycota maize pathogen Ustilago maydis, Dtps2 deletion mutants were constructed. These mutants did not produce trehalose as confirmed by HPLC analysis, showing that the single gene disruption impaired its biosynthesis. The mutants displayed increased sensitivity to oxidative, heat, acid, ionic and osmotic stresses as compared to the wild-type strains. Virulence of Dtps2 mutants to maize plants was extremely reduced compared to wild-type strains, possibly due to reduced capability to deal with the hostile host environment. The phenotypic traits displayed by Dtps2 strains were fully restored to wild-type levels when complemented with the endogenous UmTPS2 gene, or a chimeric construct having the S. cerevisiae TPS2 open reading frame. This report demonstrates the presence of a single biosynthetic pathway for trehalose, and its importance for virulence in this model Basidiomycota plant pathogen.

 

Bakkeren et al. 2016. Genomics research on non-model plant pathogens: delivering novel insights into rust fungus biology. Front. Plant Sci. 5: 216. Editorial to Frontiers E-book (http://journal.frontiersin.org/researchtopic/1925/genomics-research-on-non-model-plant-pathogens-delivering-novel-insights-into-rust-fungus-biology), covering 14 published papers for which these 3 authors were co-editors. DOI:10.3389/fpls.2016.00216

 

Segovia  et al. 2016. Two small secreted proteins from Puccinia triticina induce reduction of ß-glucoronidase transient expression in wheat isolines containing Lr9, Lr24, and Lr26. Can. J. Plant Pathol. 38(1): 91-102. DOI:10.1080/07060661.2016.1150884

Abstract: Little is known about the molecular interaction of wheat and leaf rust (Puccinia triticina Eriks). However, genomic tools are now becoming available so that the host-pathogen interactions can be better understood. Significant efforts are being placed on understanding the secretomes of various pathogens as secreted peptides are believed to be the best candidates for avirulence effectors. In this work, a P. triticina haustorial cDNA library was evaluated for the presence of proteins containing secretion signals. Ten predicted proteins were found in the library, of which two were expressed in haustorial fractions. Three of the secreted proteins, Pt3, Pt12, and Pt27, were used in biolistic experiments to determine whether they could induce hypersensitive cell death, which is commonly observed in incompatible rust interactions with wheat leaf rust resistance genes. When Pt3 was co-bombarded with a ?-glucoronidase (GUS)-expressing vector into wheat isolines with resistance genes Lr9 or Lr24, a significant reduction of GUS expression was observed, presumably due to hypersensitive cell death. In other co-bombardment experiments, Pt27 induced a significant reduction in GUS expression in the Lr26 isoline. These results suggest that Pt3 and Pt27 may function in avirulence against wheat leaf rust in resistant genotypes.

 

McCallum et al. 2016. A review of wheat leaf rust research and the development of resistant cultivars in Canada. Can. J. Plant Pathol. 38(1), 1-18. DOI:10.1080/07060661.2016.1145598.

Abstract: Wheat leaf rust, caused by Puccinia triticina Eriks., is of worldwide concern for wheat producers. The disease has been an annual problem for Canadian wheat producers since the early days of wheat cultivation in the 1800s, and research focused on combating this disease began in the early 1900s. Significant progress was made towards understanding the epidemiology of wheat leaf rust and developing genetic resistance in many countries worldwide. This review paper focuses exclusively on the research and development done in whole, or in part, in Canada. An integrated approach to controlling wheat leaf rust consisted of research in the following areas: the early research on wheat leaf rust in Canada, breeding and commercialization of high quality rust resistant wheat cultivars, discovery and genetic analysis of leaf rust resistance genes, the population biology and genetics of the P. triticina/wheat interaction. This review summarizes the research in each of these areas and the connections between the different aspects of the research. A multi-disciplinary team approach has been key to the advancements made within these diverse research fields in Canada since the early 1900s.

 

Ali, S. and Bakkeren, G. 2015. Conversion of BAC clones into binary BAC (BIBAC) vectors and their delivery into basidiomycete fungal cells using Agrobacterium tumefaciens. Bacterial Artificial Chromosomes. Ed. K. Narayanan. Clifton, N.J., Springer New York. Chapter 9: 199-215

Abstract: The genetic transformation of certain organisms, required for gene function analysis or complementation, is often not very efficient, especially when dealing with large gene constructs or genomic fragments. We have adapted the natural DNA transfer mechanism from the soil pathogenic bacterium Agrobacterium tumefaciens, to deliver intact large DNA constructs to basidiomycete fungi of the genus Ustilago where they stably integrated into their genome. To this end, Bacterial Artificial Chromosome (BAC) clones containing large fungal genomic DNA fragments were converted via a Lambda phage-based recombineering step to Agrobacterium transfer-competent binary vectors (BIBACs) with a Ustilago-specific selection marker. The fungal genomic DNA fragment was subsequently successfully delivered as T-DNA through Agrobacterium-mediated transformation into Ustilago species where an intact copy stably integrated into the genome. By modifying the recombineering vector, this method can theoretically be adapted for many different fungi.

 

Panwar et al. 2015. A functional genomics method for assaying gene function in phytopathogenic fungi through host-induced gene silencing mediated by Agroinfiltration. In: Plant Gene Silencing: Methods and Protocols. Eds. Mysore, K. S. and Muthappa, S-K. Springer New York. Chapter 13: 179-189. DOI:10.1007/978-1-4939-2453-0

Abstract: With the rapid growth of genomic information, there is an increasing demand for efficient analysis tools to study the function of predicted genes coded in genomes. Agroinfiltration, the delivery of gene constructs into plant cells by Agrobacterium tumefaciens infiltrated into leaves, is one such versatile, simple, and rapid technique that is increasingly used for transient gene expression assay in plants. In this chapter, we focus on the use of agroinfiltration as a functional genomics research tool in molecular plant pathology. Specifically, we describe in detail its use in expressing phytopathogenic fungal gene sequences in a host plant to induce RNA silencing of corresponding genes inside the pathogen, a method which has been termed host-induced gene silencing (HIGS). We target the fungal pathogen Puccinia triticina which causes leaf rust on its wheat host, but the method is applicable to a variety of pathosystems.

 

Wang et al. 2015. Sr36- and Sr5-mediated resistance response to Puccinia graminis f. sp. tritici is associated with callose deposition in wheat guard cells, Phytopathology, 105(6): 728-737. doi : 10.1094/PHYTO-08-14-0213-R

Abstract: Race-specific resistance of wheat to Puccinia graminis f. sp. tritici is primarily posthaustorial and often involves the induction of a hypersensitive response (HR). The aim of this study was to investigate host defense responses induced in interactions between P. graminis f. sp. tritici races and wheat lines carrying different race-specific stem rust resistance (Sr) genes. In incompatible interactions between wheat lines carrying Sr36 in three genetic backgrounds (LMPG, Prelude, or W2691) and avirulent P. graminis f. sp. tritici races MCCFC or RCCDM, callose accumulated within 24 h in wheat guard cells contacted by a P. graminis f. sp. tritici appressorium, and P. graminis f. sp. tritici ingress was inhibited following appressorium formation. Accordingly, the expression of transcripts encoding a callose synthase increased in the incompatible interaction between LMPG-Sr36 and avirulent P. graminis f. sp. tritici race MCCFC. Furthermore, the inhibition of callose synthesis through the infiltration of 2-deoxy-D-glucose (DDG) increased the ability of P. graminis f. sp. tritici race MCCFC to infect LMPG-Sr36. A similar induction of callose deposition in wheat guard cells was also observed within 24 h after inoculation (hai) with avirulent P. graminis f. sp. tritici race HKCJC on LMPG-Sr5 plants. In contrast, this defense response was not induced in incompatible interactions involving Sr6, Sr24, or Sr30. Instead, the induction of an HR and cellular lignification were noted. The manifestation of the HR and cellular lignification was induced earlier (24 hai) and was more extensive in the resistance response mediated by Sr6 compared with those mediated by Sr24 or Sr30. These results indicate that the resistance mediated by Sr36 is similar to that mediated by Sr5 but different from those triggered by Sr6, Sr24, or Sr30. Resistance responses mediated by Sr5 and Sr36 are prehaustorial, and are a result of very rapid recognition of molecules derived from avirulent isolates of P. graminis f. sp. tritici, in contrast to the responses triggered in lines with Sr6, Sr24, and Sr30.

 

Ali et al. 2014. An immunity-triggering effector from the barley smut fungus Ustilago hordei resides in an Ustilaginaceae-specific cluster bearing signs of transposable element-assisted evolution. PLoS Pathog 10(7): e1004223

Abstract: Upon host infection, plant pathogens secrete suites of virulence effectors to suppress defense responses and support their own development. In certain cases, hosts evolve resistance genes that recognize such effectors or their actions to initiate defense responses. By deleting candidate genes, we identified the immune-triggering effector UhAvr1 from Ustilago hordei, a barley-infecting basidiomycete smut fungus. We show that this effector is expressed only when hyphae sense and infect barley coleoptile epidermal cells. Its presence in the fungus causes a necrotic reaction immediately upon penetration resulting in complete immunity in barley cultivars having resistance gene Ruh1. We show that fungal isolates that have mutated to change the expression of this non-crucial protein are avoiding recognition by the host, hence overcoming restriction by its immune response. In virulent isolates, transposable elements, known as genome modifiers, have separated the UhAvr1 coding region from its transcription signals. UhAvr1 is located in a larger cluster of ten effectors and is similar to clusters with more and further diversified effectors in the related maize pathogens U. maydis and Sporisorium reilianum. This study should lead us to discovering a mechanism by which this major cereal crop protects itself against this pathogen.

 

Bruce et al. 2014. Using transcription of six Puccinia triticina races to identify the effective secretome during infection of wheat. Front. Plant Sci. 4: 520

Abstract: Wheat leaf rust, caused by the basidiomycete Puccinia triticina, can cause yield losses of up to 20% in wheat producing regions. During infection, the fungus forms haustoria that secrete proteins into the plant cell and effect changes in plant transcription, metabolism and defense. It is hypothesized that new races emerge as a result of overcoming plant resistance via changes in the secreted effector proteins. To understand gene expression during infection and find genetic differences associated with races, RNA from wheat leaves infected with six different rust races, at six days post inoculation, was sequenced using Illumina. As P. triticina is an obligate biotroph, RNA from both the host and fungi were present and separated by alignment to the P. triticina genome and a wheat EST reference. A total of 222,571 rust contigs were assembled from 165 million reads. An examination of the resulting contigs revealed 532 predicted secreted proteins among the transcripts. Of these, 456 were found in all races. Fifteen genes were found with amino acid changes, corresponding to putative avirulence effectors potentially recognized by 11 different leaf rust resistance (Lr) genes. Thirteen of the potential avirulence effectors have no homology to known genes. One gene had significant similarity to cerato-platanin, a known fungal elicitor, and another showed similarity to fungal tyrosinase, an enzyme involved in melanin synthesis. Temporal expression profiles were developed for these genes by qRT-PCR and show that the 15 genes share similar expression patterns from infection initiation to just prior to spore eruption.

 

Lefebvre et al. 2013. The transition from a phytopathogenic smut ancestor to an anamorphic biocontrol agent deciphered by comparative whole genome analysis. Plant Cell 25, 1946-1959. Open Access http://www.plantcell.org/cgi/content/short/tpc.113.113969?keytype=ref&ijkey=xzroSbg7ePaYOZV

Abstract: Pseudozyma flocculosa is related to the model plant pathogen Ustilago maydis yet is not a phytopathogen but rather a biocontrol agent of powdery mildews; this relationship makes it unique for the study of the evolution of plant pathogenicity factors. The P. flocculosa genome of 23 Mb includes 6877 predicted protein coding genes. Genome features, including hallmarks of pathogenicity, are very similar in P. flocculosa and U. maydis, Sporisorium reilianum, and Ustilago hordei. Furthermore, P. flocculosa, a strict anamorph, revealed conserved and seemingly intact mating-type and meiosis loci typical of Ustilaginales. By contrast, we observed the loss of a specific subset of candidate secreted effector proteins reported to influence virulence in U. maydis as the singular divergence that could explain its nonpathogenic nature. These results suggest that P. flocculosa could have once been a virulent smut fungus that lost the specific effectors necessary for host compatibility. Interestingly, the biocontrol agent appears to have acquired genes encoding secreted proteins not found in the compared Ustilaginales, including necrosis-inducing-Phytophthora-protein- and Lysin-motif- containing proteins believed to have direct relevance to its lifestyle. The genome sequence should contribute to new insights into the subtle genetic differences that can lead to drastic changes in fungal pathogen lifestyles.

 

Panwar et al. 2013. Host-generated trans-specific RNAi of wheat leaf rust fungus Puccinia triticina pathogenicity genes induced by the Barley stripe mosaic virus. Plant Mol Biol 81, 595-608

Abstract: Rust fungi are devastating plant pathogens and several Puccinia species have a large economic impact on wheat production worldwide. Disease protection, mostly offered by introgressed host-resistance genes, is often race-specific and rapidly overcome by newly-emerging virulent strains. Extensive new genomic resources have identified vital pathogenicity genes but their study is hampered because of the biotrophic life styles of rust fungi. In cereals, Barley stripe mosaic virus (BSMV)-induced RNAi has emerged as a useful tool to study loss-of-function phenotypes of candidate genes. Expression of pathogen-derived gene fragments in this system can be used to obtain in planta-generated silencing of corresponding genes inside biotrophic pathogens, a technique termed host-induced gene silencing (HIGS). Here we test the effectiveness of BSMV-mediated HIGS in the wheat leaf rust fungus Puccinia triticina (Pt) by targeting three predicted pathogenicity genes, a MAPK, a cyclophilin, and a calcineurin regulatory subunit. Inoculation of BSMV RNAi constructs generated fungal gene-specific siRNA molecules in systemic leaves of wheat plant. Subsequent Pt inoculation resulted in a suppressed disease phenotype and a reduction in endogenous transcript levels of the targeted fungal genes indicating translocation of siRNA molecules from host to fungal cells. Efficiency of this host-generated trans-specific RNAi was enhanced by using BSMV silencing vectors defective in coat protein coupled with introducing fungal gene sequences simultaneously in sense and antisense orientation. The disease suppression indicated the likely involvement of these fungal genes in pathogenicity. This study demonstrates that BSMV-mediated in planta-generated RNAi is an effective strategy for functional genomics in rust fungi.

 

Panwar et al. 2013. Endogenous silencing of Puccinia triticina pathogenicity genes through in planta-expressed sequences leads to suppression of rust diseases on wheat. Plant J 73, 521-532

Abstract: Rust fungi are destructive plant pathogens. The draft genomes of several wheat-infecting species have been released and potential pathogenicity genes identified through comparative analyses to fungal pathogens that are amenable to genetic manipulation. Functional gene analysis tools are needed to understand the infection process of these obligate parasites and to confirm whether predicted pathogenicity genes could become targets for disease control. We have modified an Agrobacterium tumefaciens-mediated in planta-induced transient gene silencing (PITGS) assay for use in wheat and used this assay to target predicted wheat leaf rust fungus, Puccinia triticina (Pt) pathogenicity genes, a MAP kinase (Pt MAPK1), a cyclophilin (Pt CYC1) and calcineurin B (Pt CNB), to analyze their role in disease. Agroinfiltration effectively delivered hairpin silencing constructs in wheat leading to the generation of fungal gene-specific siRNA molecules in infiltrated leaves and resulting in up to 70% reduction in transcription of the endogenous target genes in superinfected Pt. In vivo silencing caused severe disease suppression, compromising fungal growth and sporulation as viewed by confocal microscopy and measured by reductions in fungal biomass and emergence of uredinia. Interestingly, using the same gene constructs, suppression of infection by P. graminis and P. striiformis was also achieved. Our results show that A. tumefaciens-mediated PITGS can be used as a reverse genetics tool to discover gene function in rust fungi. This proof-of-concept study indicates that the targeted fungal transcripts might be important in pathogenesis and could potentially be utilized as promising targets for developing RNA interference-based resistance against rust fungi.

 

Fellers et al. 2013. Conserved loci of leaf and stem rust share synteny interrupted by lineage-specific influx of repeat elements. BMC Genomics 14:60

Abstract: BACKGROUND:Wheat leaf rust (Puccinia triticina Eriks; Pt) and stem rust fungi (P. graminis f.sp. tritici; Pgt) are significant economic pathogens having similar host ranges and life cycles, but different alternate hosts. The Pt genome, currently estimated at 135 Mb, is significantly larger than Pgt, at 88 Mb, but the reason for the expansion is unknown. Three genomic loci of Pt conserved proteins were characterized to gain insight into gene content, genome complexity and expansion.RESULTS:A bacterial artificial chromosome (BAC) library was made from P. triticina race 1, BBBD and probed with Pt homologs of genes encoding two predicted Pgt secreted effectors and a DNA marker mapping to a region of avirulence. Three BACs, 103 Kb, 112 Kb, and 166 Kb, were sequenced, assembled, and open reading frames were identified. Orthologous genes were identified in Pgt and local conservation of gene order (microsynteny) was observed. Pairwise protein identities ranged from 26 to 99%. One Pt BAC, containing a RAD18 ortholog, shares syntenic regions with two Pgt scaffolds, which could represent both haplotypes of Pgt. Gene sequence is diverged between the species as well as within the two haplotypes. In all three BAC clones, gene order is locally conserved, however, gene shuffling has occurred relative to Pgt. These regions are further diverged by differing insertion loci of LTR-retrotransposon, Gypsy, Copia, Mutator, and Harbinger mobile elements. Uncharacterized Pt open reading frames were also found; these proteins are high in lysine and similar to multiple proteins in Pgt.CONCLUSIONS:The three Pt loci are conserved in gene order, with a range of gene sequence divergence. Conservation of predicted haustoria expressed secreted protein genes between Pt and Pgt is extended to the more distant poplar rust, Melampsora larici-populina. The loci also reveal that genome expansion in Pt is in part due to higher occurrence of repeat-elements in this species.

 

Laurie et al. 2013. Do TE activity and counteracting genome defenses, RNAi and methylation, shape the sex lives of smut fungi? Plant Signal. Behavior 8, e23853

Abstract: The availability of three genomes from smut fungi differing in mating, TE load, and genome defense mechanisms, allowed a comparative analyses and a discussion on evolutionary forces shaping them. A complex balance of selective forces seems at play. A bipolar mating system in Ustilago hordei promotes selfing, advantageous for successful niche occupation but favoring accumulation of repetitive DNA, including TEs. TE activity may have caused genome variations necessary for these obligate parasites under high host selection pressures. Higher TE activity is balanced by genome defenses through recombination, RN Ai, methylation and RI P mutagenesis. In tetrapolar U. maydis, lacking silencing and possibly methylation mechanisms, reduced inbreeding potential favors removal of repetitive DNA, presumably by its highly-efficient recombination system.

 

Wang et al. 2013. Comparative microscopic and molecular analysis of Thatcher near-isogenic lines with wheat leaf rust resistance genes Lr2a, Lr3, LrB or Lr9 upon challenge with different Puccinia triticina races. Plant Pathology Plant Pathology 62, 698-707

Abstract: Thatcher near-isogenic lines (NILs) of wheat carrying resistance gene Lr2a, Lr3, LrB or Lr9 were inoculated with Puccinia triticina races of virulence phenotype BBBD, MBDS, SBDG and FBDJ. Puccinia triticina infection structures were analysed under the fluorescence microscope over a course of 14 days after inoculation (dai). The relative proportion of P. triticina and wheat genomic DNA in infected leaves was estimated with a semi-quantitative multiplex PCR analysis using P. triticina- and wheat-specific primers. The occurrence of a hypersensitive response (HR), cellular lignification and callose deposition in inoculated plants was investigated microscopically. In interactions producing highly resistant infection type (IT) ‘0;’, a maxi- mum of two haustorial mother cells per infection site were produced, and there was no increase in the proportion of P. triticina genomic DNA in infected leaves, indicating the absence of P. triticina growth. In comparison, sizes of P. triticina colonies increased gradually in interactions producing moderately resistant IT ‘1’ and ‘2’, with the highest proportion of P. triticina genomic DNA found in leaves sampled at 14 dai. In interactions producing susceptible IT ‘3–4’, the highest proportion of P. triticina genomic DNA was found in leaves sampled at 10 dai (45.5–51.5%). HR and cellular lignification were induced in interactions producing IT ‘0;’ and ‘1’ at 1 dai but they were not observed in interactions producing IT ‘2’ until 2 dai. No HR or cellular lignification were induced in interactions producing susceptible IT ‘3–4’. Furthermore, a strong deposition of callose was induced in Lr9 + BBBD and Lr9 + FBDJ (IT ‘0;’), whereas this defence response was not induced in resistant or susceptible interactions involving Lr2a, Lr3 or LrB, indicating that Lr9 mediated resistance was different from that conditioned by Lr2a, Lr3 or LrB.

 

Laurie, J. D. et al. 2012. Genome comparison of barley and maize smut fungi reveals targeted loss of RNA silencing components and species-specific presence of TEs. Plant Cell 24, 1733-1745

Abstract: Ustilago hordei is a biotrophic parasite of barley (Hordeum vulgare). After seedling infection, the fungus persists in the plant until head emergence when fungal spores develop and are released from sori formed at kernel positions. The 26.1-Mb U. hordei genome contains 7113 protein encoding genes with high synteny to the smaller genomes of the related, maize-infecting smut fungi Ustilago maydis and Sporisorium reilianum but has a larger repeat content that affected genome evolution at important loci, including mating-type and effector loci. The U. hordei genome encodes components involved in RNA interference and heterochromatin formation, normally involved in genome defense, that are lacking in the U. maydis genome due to clean excision events. These excision events were possibly a result of former presence of repetitive DNA and of an efficient homologous recombination system in U. maydis. We found evidence of repeat-induced point mutations in the genome of U. hordei, indicating that smut fungi use different strategies to counteract the deleterious effects of repetitive DNA. The complement of U. hordei effector genes is comparable to the other two smuts but reveals differences in family expansion and clustering. The availability of the genome sequence will facilitate the identification of genes responsible for virulence and evolution of smut fungi on their respective hosts.

 

Bakkeren, G. et al. 2012. Functional genomic approaches in cereal rusts. Can J Plant Pathol. 34, 3-12

Abstract: Cereal rust fungi are pathogens of major importance to agriculture, threatening cereal production worldwide. Targeted breeding for resistance, based on information from fungal surveys and population structure analyses of virulence, has been effective. Nevertheless, breakdown of resistance occurs frequently and continued efforts are needed to understand how these fungi overcome resistance and to determine the range of available resistance genes. The development of genomic resources for these fungi and their comparison has released a torrent of new ideas and approaches to use this information to assist pathologists and agriculture in general. The sequencing of gene transcripts and the analysis of proteins from haustoria has yielded candidate virulence factors among which could be defence-triggering avirulence genes. Genome-wide computational analyses, including genetic mapping and transcript analyses by RNA sequencing of many fungal isolates, will predict many more candidates. Functional assays, such as leaf infiltration using Agrobacterium for delivery of cloned fungal effectors, are being developed. This will allow the screening of wheat germplasm for novel resistance genes for breeding. Comparative analyses have also revealed fungal virulence genes, providing fungal targets for disease control in host-produced RNAi approaches.

 

Bakkeren, G. et al. 2012. Sex and Virulence in Basidiomycete Pathogens. In: Evolution of Virulence in Eukaryotic Microbes. Eds. L. David Sibley, Barbara J. Howlett, and Joseph Heitman. Wiley-Blackwell, Hoboken, New Jersey. ISBN 978-1-118-03818-5. Pp. 437-460

Abstract: Amongst the tens of thousands of known basidiomycete fungal species is an important group that contains pathogens of humans, animals, plants and trees. Basidiomycete pathogens of humans are a growing group represented by the well-studied model organism Cryptococcus neoformans. Plant basidiomycete pathogens, such as smut, bunt and rust fungi, cause major diseases in crops and trees worldwide. The most-studied model is the corn smut fungus, Ustilago maydis. Molecular determinants of the sexual cycle, pathogenicity and virulence have been the focus of many studies in both of these organisms. Sex and pathogenicity are intricately linked in the smut fungi, and for Cryptococcus and Puccinia species, which cause rusts of cereals, sex is important to generate the variability necessary to change or adapt infection strategies to overcome host defenses. We review what is known about the molecular networks in these model organisms and explore if and how sex and virulence are linked.

 

Ali, S., and Bakkeren, G. 2012. Fungal and oomycete effectors; strategies to subdue a host. Can. J. Plant Pathol. 33, 425-446

Abstract: Molecular studies focusing on the interface between microbes and plant hosts have provided major insights into the basis underlying pathogenesis, symbiosis and plant defence and resistance mechanisms. A more recent focus on microbes, facilitated by the generation of complete genome sequences, has uncovered the sheer number of protein effectors microbes deliver in this interface as well as inside host cells to manipulate the plant immune system. Although studies on the characterization and roles of bacterial effectors are further advanced, in this review we focus on the current knowledge of fungal and oomycete effectors and their roles. Examples are given of effectors disarming plant defence enzymes, such as the apoplastic effector AVR2 from Cladosporium fulvum which inhibits the tomato defence cysteine protease. Other effectors interfere with the perception by the host of microbes exposing molecular determinants such as Phytophthora infestans INF1 protein. Many effectors alter gene expression induced by the host during defence, exemplified in fungi by Ustilago maydis Pit2 suppressing maize defence genes. Effectors recognized by resistance gene products, either directly or indirectly, and eliciting defence, represent the classical avirulence genes and almost 50 have now been cloned from fungi and oomycetes. Evolutionary adaptations and arms races have produced diversification in both pathogen and host, and in pathogens, are the cause of breaking crop resistance in agricultural settings. Molecular insight provides valuable information for applications. For example, some effectors are crucial for pathogenesis, thereby revealing targets for disease control and others interact with host resistance gene products and could be used to screen germplasm for novel sources of disease resistance. Variation among effectors will likely yield diagnostic tools for pathogen race identification. The study of model systems is providing insight into avenues by which other, major plant diseases can potentially be controlled.

 

Xu et al. 2011. Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi. BMC Genomics In press

Abstract:  Background: Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology.

Results: To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with a previously known 25,558 ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to genomic sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs.

Conclusions: The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence.

 

Song et al. 2011. Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria. Proteomics 11, 944-963

Abstract:  Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world-wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2D-PAGE MS/MS and GeLC-MS were used to separate proteins. Generated spectra were compared to a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, P. graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative reverse transcription PCR, all were shown to be strongly upregulated and four expressed solely upon infection.

 

Cervantes-Chávez et al. 2011. Response to environmental stresses, cell wall integrity and virulence are orchestrated through the calcineurin pathway in Ustilago hordei. Mol Plant-Microbe Interact 24, 219-232

Abstract:  In eukaryotes, several biological processes are regulated through calcium signaling. Calcineurin is a calcium-calmodulin regulated serine/threonine phosphatase consisting of a catalytic subunit A and regulatory subunit B. Phosphatase activity resides in the catalytic subunit which activates by dephosphorylation downstream components such as transcription factor Crz1. The importance of this pathway to respond to environmental stress has been explored in several fungal pathogens. The basidiomycete Ustilago hordei causes covered smut of barley. We addressed the role of the Ca2+-calcineurin activated pathway by deleting UhCna1 and UhCnb1. These genes were not essential in U. hordei but the corresponding mutants displayed a variety of phenotypes when applying environmental stress such as sensitivity to pH, temperature, H2O2, mono- and divalent cations, and to genotoxic, acid or oxidative stresses. Cell wall integrity was compromised and mutants displayed altered cell morphologies. Mating was delayed but not abolished and combined sensitivities likely explained a severely reduced virulence towards barley plants. Expression analyses revealed that response to salt stress involved the induction of membrane ATPase genes UhEna1 and UhEna2 which were regulated through the calcineurin pathway. Upregulation of UhFKS1, a 1,3-beta-D-glucan synthase gene, correlated with the increased amount of 1,3-beta-D-glucan in the calcineurin mutants grown under salt stress.

 

Ali and Bakkeren. 2011. Introduction of large DNA inserts into the barley pathogenic fungus, Ustilago hordei, via recombined binary BAC vectors and Agrobacterium-mediated transformation. Curr Genet 57, 63-73

Abstract:  Genetic transformation of organisms with large genome fragments containing complete genes, with regulatory elements or clusters of genes, can contribute to the functional analysis of such genes. However, large inserts, such as those found on bacterial artificial chromosome (BAC) clones, are often not easy to transfer. We exploited an existing technique to convert BAC clones, containing genomic DNA fragments from the barley-covered smut fungus Ustilago hordei to binary BACs (BIBACs) to make them transferable by the Agrobacterium tumefaciens T-DNA transfer machinery. Genetic transformation of U. hordei with BAC clones using polyethylene glycol or electroporation is difficult. As a proof of concept, two BAC clones were successfully converted into BIBAC vectors and transferred by A. tumefaciens into U. hordei and U. maydis, the related corn smut fungi. Molecular analysis of the transformants showed that the T-DNA containing the BAC clones with their inserts was stably integrated into the U. hordei genome. A transformation frequency of approximately 10−4 was achieved both for U. hordei sporidia and protoplasts; the efficiencies were 25–30 times higher for U. maydis. The combination of in vivo recombineering technology for BAC clones and A. tumefaciens-mediated transformation of Ustilago species should pave the way for functional genomics studies.

 

Gaudet et al. 2010. Morphological and molecular analyses of host and nonhost interactions involving barley and wheat and the covered smut pathogen Ustilago hordei. Mol Plant-Microbe Interact 23, 1619-1634

Abstract:  Ustilago hordei interactions on coleoptiles of barley host cultivars Odessa (compatible), Hannchen (incompatible, carrying the Ruh1 resistance gene), and on nonhost Neepawa wheat were studied using light and fluorescent microscopy. Autofluorescence, mainly caused by callose accumulation, was more rapidly expressed in nonhost wheat at 30 to 72 h compared with the incompatible reaction between 72 and 144 h. Microarray results demonstrated that more than half of the 893 differentially regulated genes were observed in Neepawa; of these genes, 45% fell into the defense- and stress-related classes in Neepawa compared with 25 and 37% in Odessa and Hannchen, respectively. Their expression coincided with the early morphological defense responses observed and were associated with the jasmonic acid and ethylene (JA/ET) signaling pathway. Expression patterns in Odessa and Hannchen were similar, involving fewer genes and coinciding with later morphological defense responses of these varieties. Although no visible hypersensitive response was apparent in Hannchen or Neepawa, specific upregulation of hypersensitivity-related proteins was observed, such as beta-VPE at 48 h. Expression levels of the callose synthase gene were closely associated with callose accumulation. Differential responses in defense-gene expression among disease reaction types included upregulation of PR-1.1b and downregulation of a nonspecific lipid transfer protein in the incompatible and compatible interactions, respectively. Transcript levels of EDS1 and PAD4, involved in both basal resistance and R-mediated resistance to avirulent pathogens, were up-regulated during both nonhost and Ruh1-mediated resistance. Application of methyl-jasmonate, salicylic acid and ET to leaves revealed that only PR1.1b is strongly up-regulated by all three compounds, while the majority of the defense-related genes are only slightly up-regulated by these signaling compounds.

         

Wang et al. 2010. Virulence and molecular polymorphisms of the wheat leaf rust fungus Puccinia triticina in Canada from 1997 to 2007. Botany 88, 575-589

Abstract:  Populations of Puccinia triticina, one of the casual agents of wheat leaf rust disease, in the pacific (British Columbia and Alberta), prairie (Manitoba and Saskatchewan), and eastern regions (Quebec and Ontario) of Canada from 1997 to 2007 were analyzed for virulence and genetic diversity by revealing expressed sequence tag derived simple sequence repeat (EST-SSR) polymorphisms. Since 1997, a significant shift in the virulence of P. triticina occurred across Canada. The diversity of P. triticina virulence phenotypes in Manitoba and Saskatchewan, as measured by Shannon and Simpson indexes, decreased due to the directional selection toward predominant virulence phenotypes, whereas it remained relatively constant in Quebec and Ontario. The clustering of P. triticina virulence phenotypes from 1997 to 2007 was similar to that found in previous years, and was correlated with virulence to leaf rust resistance genes Lr2a, Lr2c, and Lr17a. Distinct EST-SSR profiles were found in different groups of P. triticina virulence phenotypes based on virulence to Lr2a, Lr2c, and Lr17a. In addition, the population of P. triticina in Manitoba and Saskatchewan was different from that in Quebec and Ontario from 1997 to 2007, based on both virulence characteristics and EST-SSR genotypes.

 

Wang et al. 2010. Development of EST-derived simple sequence repeat markers for the wheat leaf rust fungus, Puccinia triticina Eriks. Can J Plant Pathol 32, 98-107

Abstract:  Gene-associated simple sequence repeat (SSR) markers were developed for Puccinia triticina through the data mining of existing EST libraries. Analysis of 7134 expressed sequence tags (ESTs) from cDNA libraries of P. triticina detected 204 EST-SSRs with a minimum of 12 repeating nucleotides. The majority of EST-SSRs contained short di- or tri-nucleotide repeats. These EST-SSRs were evaluated on 35 P. triticina isolates collected in Canada and 21 EST-SSRs were polymorphic and informative in determining intraspecific genetic diversity. A comparison of virulence and EST-SSR genotypes showed a strong correlation between virulence to Lr2a, Lr2c and Lr17a and EST-SSRs genotypes. The differentiation of the P. triticina population based on EST-SSR genotypes was comparable to that obtained with genomic SSRs, despite differences between two types of SSR markers. Eight of the 21 EST-SSRs produced the cross amplification in Puccinia coronata and Puccinia graminis, suggesting that EST-SSRs are more applicable than genomic SSRs for interspecific analysis. In summary, our study suggests that the data mining of EST databases is a feasible way to generate informative molecular markers for genetic studies of P. triticina.

 

Bakkeren, G. et al, 2008. Sex in smut fungi: structure, function and evolution of mating type complexes. Review; special Fungal Genome issue. Fungal Genet Biol 45, S15-S21

Abstract: Smut fungi are basidiomycete plant pathogens that pose a threat to many important cereal crops. In order to be pathogenic on plants, smut fungal cells of compatible mating-type need to fuse. Fusion and pathogenicity are regulated by two loci, a and b, which harbor conserved genes. The functions of the encoded mating-type complexes have been well-studied in the model fungus Ustilago maydis and will be briefly reviewed here. Sequence comparison of the mating-type loci of different smut and related fungi has revealed that these loci differ substantially in structure. These structural differences point to an evolution from tetrapolar to bipolar mating behavior, which might have occurred several independent times during fungal speciation.

 

Laurie et al, 2008. Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis. Current Genetics 53, 49-58

Abstract:  RNA interference (RNAi) acts through transcriptional and post-transcriptional gene silencing of homologous sequences.  With the goal of using RNAi as a tool for studying gene function in the related basidiomycete cereal pathogens Ustilago hordei and U. maydis, we developed a general purpose RNAi expression vector.  Tandem, inverted fragments of the GUS gene were inserted into this vector flanking an intron and used to transform engineered GUS-expressing haploid cells.  Down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei, even though corresponding GUS double-stranded RNA was detected in both species.  Similarly, when the endogenous bW mating-type gene was targeted by RNAi, mating was reduced only in U. hordei.  Our work demonstrates the feasibility of using RNAi in U. hordei and provides experimental support for the observed lack of RNAi components in the U. maydis genome.  We hypothesize that the sharply limited transposon complement in U. maydis is a biological consequence of this absence.

 

Bakkeren et al, 2008. Sex in smut fungi: structure, function and evolution of mating type complexes. Review; special fungal genome issue. Fungal Genet Biol 45, S15-S21

Abstract:  Smut fungi are basidiomycete plant pathogens that pose a threat to many important cereal crops. In order to be pathogenic on plants, smut fungal cells of compatible mating-type need to fuse. Fusion and pathogenicity are regulated by two loci, a and b, which harbor conserved genes. The functions of the encoded mating-type complexes have been well-studied in the model fungus Ustilago maydis and will be briefly reviewed here. Sequence comparison of the mating-type loci of different smut and related fungi has revealed that these loci differ substantially in structure. These structural differences point to an evolution from tetrapolar to bipolar mating behavior, which might have occurred several independent times during fungal speciation.

 

Grewal et al, 2008. Identification of barley covered smut resistance genes and mapping of Ruh1 using Ustilago hordei strains with defined avirulence genes. Can J Plant Pathol. 30(2), 277-284

Abstract:  Covered smut of barley (Hordeum vulgare), caused by Ustilago hordei, is a seed-borne disease. To identify and map disease resistance genes, current Canadian barley cultivars, parents of six barley mapping populations, and four differentials namely ‘Hannchen’ (Ruh1), ‘Excelsior’ (Ruh2), ‘Plush’ (Ruh6), and ‘Odessa’ (universal susceptible), were evaluated. Six mated combinations of U. hordei sporidial lines harbouring known alleles of virulence (avr) and avirulence (Avr) genes were used in the evaluation. Most of the barley cultivars carried the Ruh1 resistance gene, but some also had Ruh2 or Ruh6. Many cultivars showed resistance to a U. hordei sporidial mating of genotype (avr1/avr1 avr2/avr2 avr6/avr6), virulent on ‘Hannchen’, ‘Excelsior’, and ‘Plush’. The resistance indicated the presence of novel avirulence gene(s) corresponding to a postulated resistance gene(s) in these cultivars. Fifty-six doubled-haploid lines from the mapping population ‘Harrington’/TR306 were inoculated with a sporidial mating of genotype SM1 (Avr1/Avr1 avr2/avr2 avr6/avr6), avirulent on ‘Hannchen’ (Ruh1) but virulent on ‘Excelsior’ (Ruh2) and ‘Plush’ (Ruh6). ‘Harrington’ (ruh1) was susceptible to this fungal genotype possessing Avr1 and TR306 (Ruh1) was resistant. Based on field and greenhouse tests, barley lines segregated 27 resistant to 29 susceptible, which was not significantly different from a 1:1 ratio (χ2 = 0.07, P = 0.79); this indicated the presence of the single major resistance gene Ruh1 in TR306. Ruh1 mapped to the short arm of chromosome 1 (7H) between markers iPgd1A and BCD129 on the ‘Harrington’/TR306 map.

 

Hu et al, 2007. Generation of a wheat leaf rust, Puccinia triticina, EST database from stage-specific cDNA libraries. Mol Plant Pathol, 8, 451-467

Abstract:  Thirteen cDNA libraries constructed from small amounts of leaf rust mRNA using optimized methods, served as the source for the generation of 25,558 high-quality DNA sequence reads.  Five life cycle stages were sampled: resting urediniospores, urediniospores germinated over water or plant extract, compatible, interactive stages during appressorium or haustorium formation just before sporulation, and an incompatible interaction.  mRNA populations were subjected to treatments such as full-length cDNA production, subtractive and normalizing hybridizations, and size selection methods combined with PCR amplification.  Pathogen and host sequences from interactive libraries were differentiated in silico using cereal and fungal sequences, codon usage analyses, and by means of a partial prototype cDNA microarray hybridized with genomic DNAs.  This yielded a non-redundant unigene set of 9,760 putative fungal sequences consisting of 6,616 singlets and 3,144 contigs, representing 4.7 M base pairs.  At E-value ≤ 10-5, 3,670 unigenes (38%) matched sequences in various databases and collections but only 694 unigenes (7%) were similar to genes with known functions.  296 unigenes were identified as most likely wheat and 10 as rRNA sequences.  Annotation rates were low for germinated urediniospores (4%) and appressoria (2%).  Gene sets obtained from the various life cycle stages appear to be remarkably different suggesting drastic reprogramming of the transcriptome during these major differentiation processes.  Redundancy within contigs yielded information about possible expression levels of certain genes among stages.  Many sequences were similar to genes from other rusts such as Uromyces and Melampsora species; some of these genes have been implicated in pathogenicity and virulence.

 

Hu et al, 2007. Complementation of Ustilago maydis MAPK mutants by a wheat leaf rust, Puccinia triticina homolog; potential for functional analyses of rust genes. Mol Plant-Microbe Interact, 20, 637-647

Abstract:  From a large EST database representing several developmental stages of P. triticina, we discovered a MAPK with homology to kinases with known pathogenic functions in other fungi.  This PtMAPK1 is similar to the Ustilago maydis MAPK, Ubc3/Kpp2, but has a longer N-terminal extension of 43 aa with identities to U. maydis Kpp6, a homolog of Ubc3/Kpp2 with a 170 aa N-terminal extension.  Ubc3/Kpp2 is involved in mating and subsequent pathogenic development whereas Kpp6 functions during invasive growth in corn tissue.  PtMAPK1, expressed from a Ustilago-specific promoter, was able to complement a ubc3/kpp2 deletion mutant and restore mating.  It also substantially increased virulence on corn, measured as tumor formation, of a kpp6 deletion mutant.  Moreover, this construct restored to near-full pathogenicity a ubc3/kpp2 kpp6 non-pathogenic double deletion mutant.  Complementation of the ubc3/kpp2 mutant with the complete PtMAPK gene and verification of expression by RT-PCR, indicated that the rust promoter is recognized in U. maydis.  Phylogenetically, these basidiomycete plant pathogens are related, which was reflected in comparison of Pt ESTs to U. maydis gene sequences.  The U. maydis heterologous expression system allows functional analysis of rust genes, currently frustrated by the lack of efficient transformation and selection procedures.

  

Bakkeren et al, 2006. Physical mapping of the genome of the fungal pathogen Ustilago hordei and annotation of the 500 kb MAT-1 sequence. Fungal Genet Biol 43, 655-666

Abstract:  Sex in basidiomycete fungi is controlled by tetrapolar mating systems in which two unlinked gene complexes determine up to thousands of mating specificities, or by bipolar systems in which a single locus (MAT) specifies different sexes. The genus Ustilago contains bipolar (Ustilago hordei) and tetrapolar (Ustilago maydis) species and sexual development is associated with infection of cereal hosts. The U. hordei MAT-1 locus is unusually large (approximately 500 kb) and recombination is suppressed in this region. We mapped the genome of U. hordei and sequenced the MAT-1 region to allow a comparison with mating-type regions in U. maydis. Additionally the rDNA cluster in the U. hordei genome was identified and characterized. At MAT-1, we found 47 genes along with a striking accumulation of retrotransposons and repetitive DNA; the latter features were notably absent from the corresponding U. maydis regions. The tetrapolar mating system may be ancestral and differences in pathogenic life style and potential for inbreeding may have contributed to genome evolution.

 

Naik et al, 2006. Development and linkage mapping of E-STS and RGA markers for functional gene homologues in apple. Genome, 49, 959-968

Abstract:  Linkage maps developed from known-function genes can be valuable in the candidate gene mapping approach. A set of 121 expressed sequence tagged site (E-STS) primer pairs were tested on a framework genetic linkage map of apple (Malus × domestica Borkh.) constructed using simple sequence repeats (SSRs) and randomly amplified polymorphic DNA (RAPD) markers. These known-function gene markers, E-STSs, were supplemented by markers for resistance gene analogues (RGAs), designed based on conserved motifs in all characterized resistance genes isolated from plant species. A total of 229 markers, including 46 apple E-STSs, 8 RGAs, 85 SSRs from apple and peach, and 88 RAPDs, were assigned to 17 linkage groups covering 832 cM of the apple genome based on 52 individuals originating from the cross ‘Antonovka debnicka’ (Q12-4) × ‘Summerred’.  Clusters of E-STS and RGA loci were located in linkage groups previously identified to carry resistance genes, some of which confer resistance to apple scab disease caused by Venturia inaequalis (Cke.) Wint.

 

Webb et al, 2006. Transient Expression and insertional mutagenesis of Puccinia triticina using biolistics. Funct Int Genomics, 6, 250-260

Abstract:  The fungal genus Puccinia contains more than 4,000 species. Puccinia triticina, causal agent of wheat leaf rust, is an economically significant, biotrophic basidiomycete. Little is known about the molecular biology of this group, and tools for understanding gene function have not yet been established. A set of parameters was established for the transient transformation of urediniospores. The expression of three heterologous promoters (actin, elongation factor 1-alpha, and Hss1, Heat Shock 70 protein), derived from Puccinia graminis, was evaluated along with the potential for insertional mutagenesis. The UidA (GUS) gene was used as a marker for transient expression. When transferred into P. triticina urediniospores, transient expression was observed across four helium pressures using one size of gold and three sizes of tungsten microprojectiles. Each of the three promoters displayed strong transient expression in germinated urediniospores; however, higher numbers of GUS-positive urediniospores were observed when either the actin or Hss1 promoters were used. Possible concomitant insertional mutagenesis of several avirulence genes was selected in wheat cultivars harboring the cognate resistance genes. Using a linearized cloning plasmid, stable integration into the genome was achieved as demonstrated by PCR and sequencing analysis.

 

Linning et al, 2004. Marker-based cloning of the region containing the UhAvr1 avirulence gene from the basidiomycete barley pathogen Ustilago hordei. Genetics 166, 99 -111

Abstract: Race-cultivar specialization during the interaction of the basidiomycete smut pathogen Ustilago hordei with its barley host was described in the 1940's. Subsequent genetic analyses revealed the presence of dominant avirulence genes in the pathogen that conform to the gene-for-gene theory. This pathosystem therefore presents an opportunity for the molecular genetic characterization of fungal genes controlling avirulence. We performed a cross between U. hordei strains to obtain fifty-four progeny segregating for three dominant avirulence genes on three differential barley cultivars. Bulked segregant analysis was used to identify RAPD and AFLP markers tightly linked to the avirulence gene UhAvr1. The UhAvr1 gene is located in an area containing repetitive DNA and this region is undetectable in cosmid libraries prepared from the avirulent parental strain. PCR and hybridization probes developed from the linked markers were therefore used to identify cosmid clones from the virulent (Uhavr1) parent. By walking on Uhavr1-linked cosmid clones, a non-repetitive, nearby probe was found that recognized five overlapping BAC clones spanning 170 kb from the UhAvr1 parent. A contig of the clones in the UhAvr1 region was constructed and selected probes were used for RFLP analysis of the segregating population. This approach genetically defined an approximately 80 kb-region that carries the UhAvr1 gene and provided cloned sequences for subsequent genetic analysis. UhAvr1 represents the first avirulence gene cloned from a basidiomycete plant pathogen.

 

Braithwaite et al, 2004. Genetic variation in a worldwide collection of the sugarcane smut fungus Ustilago scitaminea. Proc Aust Soc Sugar Cane Technol, Vol. 26, 48-56

Abstract:  The sugarcane smut fungus, Ustilago scitaminea, first appeared in Australia in the Ord River Irrigation Area (ORIA) in July 1998. The most likely source of this infection was thought to be wind blown spores from Indonesia. Currently, the sugar industries of eastern Australia, Fiji and Papua New Guinea are still free of the disease. However, the risk of a smut incursion into eastern Australia is very high. Australian sugarcane cultivars are currently being screened in Indonesia and the ORIA to obtain smut resistance ratings, and results show that 70% of Australian cultivars are susceptible. As the use of resistant cultivars is the best option for long-term control of smut, a high priority of Australian sugarcane breeding programs is to increase the level of smut resistance in commercial cultivars. However, successful disease control requires an understanding of the level of diversity in the pathogen population. Information on the smut pathogen present in Australia and neighbouring sugar industries will enable plant breeders to select appropriate breeding strategies, including germplasm selection, for increased resistance in Australian sugarcane. The DNA fingerprinting technique of amplified fragment length polymorphisms (AFLPs) was used to assess genetic variation between isolates of the sugarcane smut fungus. The fungal collection comprised 38 isolates from 13 countries with some isolates collected from the same sugar industry 15 years apart. The technique revealed a low level of variation at the genomic DNA level, but a divergent group of isolates from Southeast Asia was identified. Sugarcane smut spores from this region could show different virulence patterns on Australian cultivars and could constitute another incursion threat to the Australian sugar industry.

 

Hu et al, 2003. Ultrastructural comparison of a compatible and incompatible interaction triggered by the presence of an avirulence gene during early infection of the smut fungus, Ustilago hordei, in barley. Physiological Mol Plant Pathol. 62, 155-166

Abstract: Cell morphologies and reactions during infection of barley by the smut fungus, Ustilago hordei, were investigated by TEM. We compared compatible and incompatible interactions caused respectively by the absence or presence of an avirulence gene (V1) on cultivar 'Hannchen', harboring the cognate resistance gene (Ruh1). In both interactions, U. hordei penetrated coleoptile epidermal cells directly within two days post inoculation (dpi). Upon penetration, an electron-opaque interfacial matrix formed around both inter- and intracellular hyphae in compatible interactions. Hyphae grew and extended into the host bundle sheath and invaded parenchyma cells. At 12 dpi, cell wall appositions formed surrounding the hyphae. Tubule-like structures of variable thickness and orientation were visible in the interfacial matrix mainly in the outer region bordering these appositions. In contrast, in incompatible interactions, cell wall appositions occurred as soon as hyphae penetrated host epidermal cells. Material, thicker and more granular in appearance compared to that in compatible interactions, was deposited around the invading hyphae and extended onto the inner surface of invaded epidermal cells. Upon penetration, host cell reactions included disorganization of cytoplasm and organelles leading to necrosis and cell death. This gene-for-gene combination triggers a very early hypersensitive response like resistance reaction, extremely localized at sites of primary infection which involves only a few plant cells and may or may not be responsible for fungal arrest.

 

Menzies et al, 2003. The use of inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphisms (AFLPs) to analyze genetic relationships among species of Ustilago. Phytopathology 93, 167-175

Abstract: In the smut fungi, few features are available for use as taxonomic criteria (spore size, shape, morphology, germination type and host range). The use of DNA-based molecular techniques are useful in expanding the traits considered in determining relationships among these fungi. We examined the phylogenetic relationships among seven species of Ustilago (U. avenae, U. bullata, U. hordei, U. kolleri, U. nigra, U. nuda, and U. tritici) using ISSRs and AFLPs to compare their DNA profiles. Fifty-four isolates of different Ustilago species were analyzed using ISSR primers, and 16 isolates of Ustilago were studied using AFLP primers. The variability among isolates within species was low for all species except U. bullata. The isolates of U. bullata, U. nuda, and U. tritici were well separated and our data supports their speciation. Ustilago avenae and U. kolleri isolates did not separate from each other and there was little variability between these species. Ustilago hordei and U. nigra isolates also showed little variability between species, but the isolates from each species grouped together. Our data suggest that U. avenae and U. kolleri are monophyletic and should be considered one species, as are U. hordei and U. nigra.

 

Hu et al, 2002. Sporidial mating and infection process of the smut fungus, Ustilago hordei, in susceptible barley. Can J Bot, 80, 1103-1114

Abstract: Ustilago hordei (Pers.) Lagerh. causes covered smut of barley and oats. Sporidial mating and the infection process on compatible barley plants, cv. Hannchen, were investigated using light microscopy and scanning and transmission electron microscopy. Within 2 h after mixing of sporidia of opposite mating types on water agar, polar conjugation tubes emerged that subsequently fused, producing infection hyphae at the junctions. Similar events occurred on germinated barley shoots, although sporidia regularly produced several conjugation tubes, of which only one was involved in mating. Tubes emerging from the sides of cells were also observed. Infection hyphae emerged from either the conjugation tube or conjugated cell body. Hyphae elongated along the shoot surface until characteristic crook and appressorium-like structures were formed. An invading hypha emerged beneath this structure and directly penetrated the underlying epidermal cell. Hyphae extended both intra- and inter-cellularly into tissues, without much branching, before becoming established in the shoot meristematic region. Plant plasma membranes remained intact during pathogen ingress and an electron-dense matrix of unknown origin appeared in the interface between plant plasma membrane and invading hypha. A large fungal biomass was generated in the host spike tissue at 42–63 days postinoculation during the development of the floral meristem.

 

Zhou et al, 2002. Genetic analysis and discrimination of sweet cherry cultivars using amplified fragment length polymorphism fingerprints. J Amer Soc Hort Sci, 127, 786-792

Abstract: Amplified fragment length polymorphisms (AFLPs) were used to analyze the relationships between sweet cherry (Prunus avium L.) cultivars and selections from the breeding program at the Pacific Agri-Food Research Centre in Summerland, Canada. Six pairs of preselected primers were used for the analysis of a total of 67 cultivars and selections. Scoring the absence and presence of 118 polymorphic DNA fragments produced a unique binary code for each cultivar and selection. Two phylogenetic trees were constructed using these 118 polymorphic fragments, one tree for 55 related cultivars and selections from the Summerland breeding program and the other for 23 self incompatible cultivars of differing origins. The reliability of AFLP DNA fingerprints was confirmed by correlating relationships revealed by AFLP profiles with known genetic relationships of some sweet cherry cultivars and by a blind test for cultivar identification. Results indicate that AFLP analysis is a good technique to evaluate genetic distance and relationships in a sweet cherry breeding population.

 

Bakkeren et al, 2000. Comparison of AFLP fingerprints and ITS sequences as phylogenetic markers in Ustilaginomycetes. Mycologia 92, 510-521

Abstract: We have compared the use of DNA sequences from the genomic internal transcribed spacer (ITS) ribosomal RNA region, with a newer method, the amplified fragment length polymorphism (AFLP) technique. ITS sequences encompass only a small part of the genome but normally reveal sufficient variability to distinguish isolates at the genus and often the species level. Although the AFLP technology reveals genome-wide restriction fragment length polymorphisms, it has not been employed extensively in establishing phylogenetic relationships. We have adapted the AFLP technology for fungal genomes and compared AFLP fingerprints generated from several fungal species and isolates from the order Ustilaginales: Ustilagohordei, U. nigra, U. aegilopsidis, U. avenae, U. kolleri, U. bullata, U. nuda, U. tritici, U. maydis, U. scitaminea, Sporisorium reilianum, and Tilletiales: Tilletia indica and T. walkeri. Geographical isolates of U. hordei and related species, particularly those infecting small-grain cereals, were difficult to distinguish when comparing ITS sequences, but were clearly separated when comparing AFLP fingerprints. The abundance of polymorphisms makes the AFLP technique more suitable to distinguish organisms in clusters of closely related species and at the isolate level. Phylogenetic analyses of the data sets generated with the two methods revealed that the AFLP-derived phylogenetic relationships were not in disagreement with the ITS-derived tree. The fungal phylogenetic tree correlated additionally with one from the graminaceous hosts generated from literature data, suggesting coevolution of some specialized host-pathogen systems. The clustering of small grain-infecting smuts due to limited genetic variability, in combination with other molecular, mating and literature data, suggests reclassification of this group possibly to include varietas designations to define host range.

 

Lee et al, 1999. The mating type and pathogenicity locus of the fungus Ustilago hordei spans a 500-kb region. Proc Natl Acad Sci USA 96, 15026-15031

Abstract: The fungal pathogen Ustilago hordei causes covered smut disease of barley and oats. Mating and pathogenicity in this fungus are controlled by the MAT locus which contains two distinct gene complexes, a and b. In this study, we tagged the a and b regions with the recognition sequence for the restriction enzyme I-SceI and determined that the distance between the complexes is 500 kb in a MAT-1 strain and 430 kb in a MAT-2 strain. Characterization of the organization of the known genes within the a and b gene complexes provided evidence for non-homology and sequence inversion between MAT-1 and MAT-2. Antibiotic resistance markers were also used to tag the a gene complex in MAT-1 strains (phleomycin-resistance) and the b gene complex in MAT-2 strains (hygromycinB-resistance). Crosses were performed with these strains and progeny resistant to both antibiotics were recovered at a low frequency suggesting that recombination is suppressed within the MAT region. Overall, the chromosome homologues carrying the U. hordei MAT locus share features with primitive sex chromosomes, with the added twist that the MAT locus also controls pathogenicity.

 

Bakkeren et al, 1996. The pheromone cell signalling components of the Ustilago a mating-type loci determine intercompatibility between species. Genetics 143, 1601-1613

Abstract: The MAT region of Ustilago hordei, a bipolar barley pathogen, harbors distinct mating functions (a and b loci). Here, we show that the b locus is essential for mating and pathogenicity, and can induce pathogenicity when introduced into a strain carrying a b locus of opposite specificity. Transformation experiments using components of the a1 locus and analysis of resulting dual mating phenotypes revealed that this locus harbors a pheromone receptor gene (Uhpra1) and a pheromone gene (Uhmfa1). These U. hordei a1 genes, when introduced by transformation, are necessary and sufficient to make U. maydis, a tetrapolar corn pathogen, intercompatible with U. hordei MAT-2, but not MAT-1, strains. U. hordei strains transformed with the U. maydis a1 locus also become intercompatible with U. maydis a2, but not a1, strains. The interspecies hybrids produced dikaryotic hyphae but were not fully virulent on either corn or barley. Partial, natural intercompatibility was shown to exist between the sugarcane smut U. scitaminea and both U. hordei and U. maydis. These results show that the signal transduction pathway for mating responses is conserved between different smut species. We conclude that, apart from intraspecies compatibility, the Ustilago a locus also dictates intercompatibility in this group of fungi.

 

Bakkeren and Kronstad, 1994. Linkage of Mating-Type Loci Distinguishes Bipolar from Tetrapolar Mating in Basidiomycetous Smut Fungi. Proc Natl Acad Sci USA, 91, 7085-7089

Abstract: Sexual compatibility requires self vs. non-self recognition. Genetically, two compatibility or mating-type systems govern recognition in heterothallic basidiomycete fungi such as the edible and woodrotting mushrooms and the economically important rust and smut phytopathogens. A bipolar system is defined by a single genetic locus (MAT) that can have two or multiple alleles. A tetrapolar system has two loci, each with two or more specificities. We have employed two species from the genus Ustilago (smut fungi) to discover a molecular explanation for the genetic difference in mating systems. Ustilago maydis, a tetrapolar species, has two genetically unlinked loci that encode the distinct mating functions of cell fusion (a locus) and subsequent sexual development and pathogenicity (b locus). We have recently described a b locus in a bipolar species, Ustilago hordei, wherein the existence of an a locus has been suspected, but not demonstrated. We report here the cloning of an allele of the a locus (a1) from U. hordei and the discovery that physical linkage of the a and b loci in this bipolar fungus accounts for the distinct mating system. Linkage establishes a large complex MAT locus in U. hordei; this locus appears to be in a region suppressed for recombination.

 

Gold et al, 1994 Three selectable markers for transformation of Ustilago maydis. Gene 142, 225-230

Abstract: Although Ustilago maydis is readily amenable to molecular genetic experimentation, few antibiotic-resistance markers are available for DNA-mediated transformation. This poses constraints on experiments involving targeted gene disruption and complementation. To address this problem, we constructed vectors using one of three additional genes as dominant selectable markers for transformation. Two genes, sat-1 (encoding streptothricin acetyltransferase) and Sh-ble (encoding a phleomycin-resistance polypeptide). are of bacterial origin and have been engineered for expression in Ustilago sp. The third gene encodes an allele of U. maydis beta-tubulin that confers resistance to the fungicide benomyl.

 

Bakkeren and Kronstad, 1993. Conservation of the b Mating-Type Gene Complex among Bipolar and Tetrapolar Smut Fungi. Plant Cell 5, 123-136

Abstract: In the phytopathogenic fungus Ustilago hordei, one locus with two alternate alleles, MAT-1 and MAT-2, controls mating and the establishment of the infectious dikaryon (bipolar mating). In contrast for U. maydis, these functions are associated with two different gene complexes, called a and b (tetrapolar mating); the a complex has two alternate specificities, and the b gene complex is multiallelic. We have found homologs for the b gene complex in U. hordei and have cloned one from each mating type using sequences from one bEast allele of U. maydis as a probe. Sequence analysis revealed two divergent open reading frames in each b complex, which we called bW (bWest) and bE (bEast) in analogy with the b gene complex of U. maydis. The predicted bW and bE gene products from the two different mating types showed apprx 75% identity when homologous polypeptides were compared. All of the characterized bW and bE gene products have variable amino-terminal regions, conserved carboxy-terminal regions, and similar homeodomain motifs. Sequence comparisons with the bW1 and bE1 genes of U. maydis showed conservation in organization and structure. Transformation of the U. hordei b gene complex into a U. hordei strain of opposite mating type showed that the b genes from the two mating types are functional alleles. The U. hordei b genes, when introduced into U. maydis, rendered the haploid transformants weakly pathogenic on maize. These results indicate that structurally and functionally conserved b genes are present in U. hordei.

 

Bakkeren et al, 1992. The a and b loci of Ustilago maydis hybridize with DNA sequences from other smut fungi. Mol Plant-Microbe Interact, 5, 347-355

Abstract: The smut fungi are obligately parasitic during the sexual phase of their life cycle, and the mating-types genes of these fungi play key roles in both sexual development and pathogenicity. Among species of smut fungi it is common to find a bipolar mating system in which one locus with two alternate alleles is believed to control cell fusion and establishment of the infectious cell type. Alternatively, several species have a tetrapolar mating systemin which two different genetic loci, one of which has multiple alleles, control fusion and subsequent development of the infection hypae. Cloned subsequences from the a and b mating-type loci of the tetrapolar smut fungus Ustilago maydis were used as hybridization probes to DNAs from 23 different fungal strains, including smut fungi with both tetrapolar and bipolar mating systems. In general, all of the smut fungi hybridized with the mating-type genes from U. maydis, suggesting conservation of the sequences involved in mating interactions. A selection of DNAs from other ascomycete and basidiomycete fungi failed to hybridize with the U. maydis mating-type sequences. Exceptions to the findings include hybridization of DNA from the a1 idiomorph of U. maydis to DNA from one strain of U. violacea and hybridization of both a idiomorphs to DNA from Saccharomyces cerevisiase.

 

Mayerhofer et al, 1991. T-DNA integration: a mode of illegitimate recombination in plants. EMBO J, 10, 697-704

Abstract:  Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

 

Bakkeren et al, 1989. Recovery of Agrobacterium tumefaciens T-DNA molecules from whole plants early after transfer. Cell 57, 847-857

Abstract:  A system for the analysis of independent T-DNA transfer events from Agrobacterium to plants is described. The complete T-DNA except for the 25 bp border sequences was replaced by one genome of a plant virus so that upon transfer to the plant, a viable replicon is produced by circularization. Rescue of virus from such infected plants allowed analysis of DNA sequences at or close to the ends of T-DNA molecules. A rather conserved right border remnant of three nucleotides was found, whereas the sequences remaining at the left end were more variable. A point deletion in the left 25 bp sequence results in even less precise processing at the left end. In addition, many rescued T-DNA molecules carry small direct repeats between the joined T-DNA ends; linear T-DNA molecules are therefore transported to the plant.

 

Hohn et al, 1989. Agrobacterium-mediated gene transfer to monocots and dicots. Genome 31, 987-993

Abstract: The interaction of the soil bacterium Agrobacterium tumefaciens with plants constitutes a unique kind of genetic flux: the bacterium transfers the T-DNA part of its Ti plasmid to plant cells where it is integrated into the genome. Possible transfer intermediates, isolated from bacteria and from plants early after transfer, are described. Agroinfection, Agrobacterium-mediated delivery of plant viral genomes, is employed to monitor early events in T-DNA transfer in dicot plants. Graminaceous monocots, so far excluded from Agrobacterium's host range because of lack of tumor formation, have been shown to be “agroinfectable”. This newly discovered interaction between grasses and the pathogen is described in terms of the efficiency of gene transfer as compared with dicot hosts, the involvement of the bacterium's virulence genes, the susceptibility of various developmental stages of the host, the implications for biotechnology, and the evolutionary aspects of this host -parasite relationship.

 

Cornelissen et al, 1985. Characteristics of Trypanosome variant antigen genes active in the tsetse fly. Nucl Acid Res, 13, 4661-4676

Abstract:  Trypanosoma brucei contains a repertoire of more than 100 different genes for Variant Surface Glycoproteins (VSGs). A small and strain-specific fraction of these genes is expressed in the salivary glands of the tsetse fly (M-genes), giving rise to metacyclic Variable Antigen Types (M-VATs). Antibodies produced in a chronic trypanosome infection initiated by syringe inoculation of bloodstream forms into mammals (i.e. against B-VATs), will react with most of the M-VATs suggesting that these B-VATs express VSG genes that are similar or identical to M-genes. We have cloned DNA complementary to the VSG mRNA of four of such B-VATs and used this to characterize the corresponding VSG genes. In three of the four VATs we find a single VSG gene hybridizing with the cDNA probe and we provide supporting evidence that this gene is expressed as an M-gene. In the bloodstream repertoire these genes appear to be activated by duplicative translocation to another telomere. In all four variants the putative M-genes are telomeric and in the three cases where the location of the genes on chromosome-sized DNA molecules could be determined, the genes were located in large DNA, whereas the majority of the telomeric VSG genes are in chromosomes less than 1000 kb. Our results are best explained by models for M-gene activation involving telomeric expression sites for these genes which are separate from those used by bloodstream forms. The implications of these results for vaccination are discussed.

 

Schetgens et al, 1984. Molecular cloning and functional characterization of Rhizobium leguminosarum structural nif -genes by site-directed transposon mutagenesis and expression in E.coli minicells. J Molec Appl Gen, 2, 406-421

Abstract:  In order to study the structural organization and regulation of the expression of the nitrogenase gene cluster in Rhizobium leguminosarum PRE we selected relevant subfragments of the sym-plasmid from clone banks by homology with R. meliloti nif-genes. Site-directed Tn5 mutagenesis was applied to a nif DH-specific clone and subsequently the transposon insertions were transferred back into the wild-type rhizobial genome by homologous recombination. Phenotypic effects of Tn5 mutations in the region of the structural nif-genes were determined by measuring acetylene reduction in nodulated plants and by immunological analysis of bacteroid-specific proteins. The localization of Tn5 insertion sites was in accordance with observed consequences: two genotypically different Tn5-induced mutations within nif D caused repression of CI alpha and beta synthesis and a strong reduction of CII production, thus resulting in a Fix- phenotype. Expression of different cloned Rhizobium DNA inserts, bearing nif K, nif D, nif H, or nif DH, was achieved in Escherichia coli minicells dependent upon the presence of a strong upstream vector promoter sequence. Gene products were identified by immunoprecipitation with specific antisera. Endogenous rhizobial transcriptional start signals in one case (nif H) seemed to be recognized at a low rate by the E. coli system; in contrast, Rhizobium ribosome binding sites for all three structural nif-genes functioned normally in minicells. The approximate location of the coding regions for nif KDH genes was determined and found to be contiguous.

 

Bakkeren and Kronstad, 2007. Bipolar and tetrapolar mating systems in the Ustilaginales. In: Sex in fungi: molecular determination and evolutionary implications, Eds. J. Heitman, J. W. Kronstad, J. Taylor, L. Casselton. American Society for Microbiology. Pp. 389-404

Abstract:  The smut fungi are attractive experimental models to investigate basidiomycete mating systems and to explore the role of mating type functions in pathogenic development.  A fascinating aspect of these fungi is that their ability to cause disease on host plants is dependent on mating interactions between haploid cells leading to formation of an infectious dikaryon.  Sex and pathogenesis are thus intimately intertwined because the infectious dikaryon requires a host for proliferation and for the eventual formation of sexual spores (teliospores).  Ustilago maydis, the corn pathogen, has emerged as the primary model for studying smut fungi and is discussed in other chapters in this book.  Here we discuss other species, U. hordei in particular, that have provided useful comparative information leading to insights into the genetic basis of bipolar versus tetrapolar mating systems in the smut fungi as a group.  We first discuss the importance of smut fungi and the interactions of these pathogens with host plants to provide context for appreciating the role of mating in disease.  We then focus on the details of the mating system in U. hordei including the structure and function of the mating type loci, the genomic organization of these elements and the sequence of the 527 kb MAT-1 locus.  Comparisons between the tetrapolar mating system in U. maydis and the bipolar system of U. hordei allowed the development of a detailed view of the genomic basis of mating system organization.  This work sets the stage of a broader examination of the interconnections between genomic organization, mating systems and pathogenesis in these fungi.

 

Horton et al, 2005. Genetics of morphogenesis in Basidiomycetes. In: Applied Mycology and Biotechnology. Vol. 5 & 6. ‘Genetics, Genomics and Bioinformatics’. (Ed. D. K. Arora) Elsevier, Dordrecht. pp 353-422

Abstract:  In this chapter, our aim is to discuss the current knowledge of the genetics of morphogenesis in basidiomycetes. We begin by outlining some features that are shared among fungi in general and those that are unique to basidiomycetes. With this background of basic fungal morphology and physiology, we focus our discussion on the genetics of morphogenesis and the fascinating biology of three broad groups of basidiomycetes: the smuts, the rusts and the mushrooms. Where the smuts and rusts are considered, there is ample discussion on pathogenesis, as these two broad groupings of fungi are particularly destructive to plants worldwide. Of course, mushrooms represent a valuable commodity in their own right. Each of these broad sections encapsulates a current assessment of the environmental cues and the genes regulating morphological change during development. Each of these three taxon-focused sections also concludes with a section discussing current trends and/or future directions.

 

Bakkeren et al, 2004. The path in fungal plant pathogenicity; many opportunities to outwit the intruders? Review. In: Genetic Engineering, Principles & Methods, Vol. 26. (Ed. J. K. Setlow), Plenum Press, New York. pp. 175 - 223

Abstract: The number of genes implicated in the infection and disease processes of phytopathogenic fungi is increasing rapidly. Forward genetic approaches have identified mutated genes that affect pathogenicity, host range, virulence and general fitness. Likewise, candidate gene approaches have been used to identify genes of interest based on homology and recently through 'comparative genomic approaches' through analysis of large EST databases and whole genome sequences. It is becoming clear that many genes of the fungal genome will be involved in the pathogen-host interaction in its broadest sense, affecting pathogenicity and the disease process in planta. By utilizing the information obtained through these studies, plants may be bred or engineered for effective disease resistance. That is, by trying to disable pathogens by hitting them where it counts. In this review (48 pages including 345 references) we focus on the current understanding of fungal plant pathogenesis at the molecular level. We also briefly discuss realized and potential disease control strategies based on this understanding.

 

Kronstad et al, 1995. Control of filamentous growth by mating and cyclic-AMP in Ustilago. Proceedings of the 5th International Mycology Congress, Can J Bot, 73, 258-265

Abstract: Mating between compatible haploid cells of the corn smut fungus, Ustilago maydis, results in a switch from budding to filamentous growth. We are analyzing the multiallelic b incompatibility locus that governs maintenance of the infectious, filamentous dikaryon. Specificity regions have been identified in the N-terminal portions of the bE and bW genes at the b locus. In addition, we have found that heterozygosity at the b locus attenuates fusion. This result suggests that b gene products may exert a negative influence on some processes, in addition to their generally recognized role in maintaining filamentous growth. The b genes have also been characterized in Ustilago hordei. This species has a bipolar mating system in which the b genes are linked to genes required for pheromone production and response to form one large mating-type region (MAT locus) with two allelic specificities. In a separate study, we have discovered that defects in adenylate cyclase result in constitutive filamentous growth and greatly reduce the virulence of U. maydis on corn seedlings. Mutations have been identified that suppress the adenylate cyclase defect and restore budding growth. Sequence analysis revealed that one of the suppressor mutations is in the gene encoding the regulatory subunit of cAMP-dependent protein kinase (PKA). Mutants altered in PKA activity have the interesting phenotype of multiple budding and frequent mislocalization of the bud site.

 

Kronstad et al, 1993. The genetics of dimorphism in the smut fungi. In: Advances in Molecular Genetics of Plant-Microbe Interactions, Vol.2, pp. 325-333

Abstract: Dimorphic growth in Ustilago maydis is regulated by mating-type loci called a and b. The b region encodes two polypeptides (bE and bW) which control the formation of the infectious dikaryon upon fusion of haploid, yeast-like cells of opposite a mating type. Given that there are at least 25 naturally occurring specificities at the b locus, and that the locus must be heterozygous to trigger filamentous growth, the molecular basis of self versus non-self recognition is of particular interest. The construction of recombinants between the b1E and b2E alleles identified a 30 to 48 amino acid region which determines specificity. In addition, hybridization and sequence analyses revealed homologs of the bE and bW genes in U. hordei, a smut thought to have only the a mating function. Genes have also been identified that may be regulated by the b locus and whose products influence cell morphology. One of these genes, called rem1, functions in the switch from yeast-like to mycelial growth.

 

Bakkeren, 1989. PhD Thesis. An analysis of interkingdom movement of genetic information by rescue of Agrobacterium tumefaciens T-DNA molecules from plants. Basel University, Switzerland.

Summary:  Agrobacterium tumefaciens, a soil-borne, plant pathogenic, gram negative bacterium is capable of genetically transforming a wide variety of plants by stable integration of a defined piece of its DNA (T-DNA or transferred DNA) into the plant genome. In the natural situation this piece of DNA contains, among others, oncogenicity-genes which upon integration in the plant cell genome, will cause a neoplastic outgrowth of the transformed cells, called 'crown gall'. The T-DNA is found on large plasmids ranging in size from 190 to 240 kb and is flanked and defined by two 25 base pairs direct, imperfect border repeats. A second part of the Ti-plasmid essential for tumor formation is the virulence or vir-region of about 35 kb lenght which is involved in the preparation, transport, and possibly integration of the T-DNA. The vir-genes are found in 7 complementation groups, virA to G, and can be transcriptionally induced by plant-wound produced substances. The Vir-region acts in trans which enables the use of the so-called 'binary vector system' with T-DNA on easily manipulable small plasmids.

Upon induction of the bacterium several T-DNA molecules can be observed separated from the Ti-plasmid due to the action of at least VirD1 and the N-terminal part of VirD2. The actual T-DNA intermediate as well as the mechanisms of its transfer to the plant cell and integration in the genome are unknown.

          A system for the analysis of independent T-DNA transfer events from A. tumefaciens to plants has been set up (Chapter II). The complete T-DNA except for the 25 bp border sequences was replaced by one genome of a plant virus so that upon transfer to the plant a viable replicon is produced by circularization. Rescue of virus from such infected plants allowed analysis of DNA sequences at or close to both ends of T-DNA molecules. We have compared those ends with their original counterparts in the Ti-plasmid. The right border end revealed a strong preservation of this repeat up to the fourth base position, a position which has been shown to be used for border repeat nicking inside A. tumefaciens. The sequences remaining at the left border end were more variable and seemed more prone to (exonucleolytic) degradation. A point deletion in the left 25 bp sequence resulted in even less precise processing at the left end. In addition, many rescued T-DNA molecules carried small direct repeats between the joined T-DNA ends which is a feature of general DNA integration. We concluded that linear T-DNA molecules are transported to the plant.

          The system allows the investigation of the role of several virulence-proteins on the plant-side of this T-DNA transfer. In Chapter III T-DNA structure and the efficiency of transfer was studied in a variety of mutant strains. VirE2 mutants showed a 1000-fold drop in efficiency compared to wild-type strains, which might be due to delivery of a reduced number of T-DNA molecules to the plant cell nucleus. Since the VirE2 protein binds single stranded DNA molecules, it might play a protective role in the transport of T-strands. A VirC1 mutant was 10- to 100-fold reduced in transfer efficiency compared to wild-type strains. Preliminary data suggest that it might lead to the generation of more variable T-DNA molecules in plants. This might mean that VirC1 is involved in T-DNA processing in A. tumefaciens. A VirD2 mutant and a virD4 mutant both showed a 10-3  to 10-4 drop in transfer efficiency using agroinfection, and a 10-5 to 10-6 fold decrease in tumor forming ability compared to wild-type strains. In addition, both mutant strains seemed to give rise to T-DNA molecules with less well conserved ends in plants. A protective function and a role during integration could be implied.