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.