Pooled effector library screening in protoplasts rapidly identifies novel Avr genes.

Crop breeding for durable disease resistance is challenging due to the rapid evolution of pathogen virulence. While progress in resistance (R) gene cloning and stacking has accelerated in recent years1-3, the identification of corresponding avirulence (Avr) genes in many pathogens is hampered by the lack of high-throughput screening options. To address this technology gap, we developed a platform for pooled library screening in plant protoplasts to allow rapid identification of interacting R-Avr pairs. We validated this platform by isolating known and novel Avr genes from wheat stem rust (Puccinia graminis f. sp. tritici) after screening a designed library of putative effectors against individual R genes. Rapid Avr gene identification provides molecular tools to understand and track pathogen virulence evolution via genotype surveillance, which in turn will lead to optimized R gene stacking and deployment strategies. This platform should be broadly applicable to many crop pathogens and could potentially be adapted for screening genes involved in other protoplast-selectable traits.

Physical separation of haplotypes in dikaryons allows benchmarking of phasing accuracy in Nanopore and HiFi assemblies with Hi-C data.

BACKGROUND: Most animals and plants have more than one set of chromosomes and package these haplotypes into a single nucleus within each cell. In contrast, many fungal species carry multiple haploid nuclei per cell. Rust fungi are such species with two nuclei (karyons) that contain a full set of haploid chromosomes each. The physical separation of haplotypes in dikaryons means that, unlike in diploids, Hi-C chromatin contacts between haplotypes are false-positive signals. RESULTS: We generate the first chromosome-scale, fully-phased assembly for the dikaryotic leaf rust fungus Puccinia triticina and compare Nanopore MinION and PacBio HiFi sequence-based assemblies. We show that false-positive Hi-C contacts between haplotypes are predominantly caused by phase switches rather than by collapsed regions or Hi-C read mis-mappings. We introduce a method for phasing of dikaryotic genomes into the two haplotypes using Hi-C contact graphs, including a phase switch correction step. In the HiFi assembly, relatively few phase switches occur, and these are predominantly located at haplotig boundaries and can be readily corrected. In contrast, phase switches are widespread throughout the Nanopore assembly. We show that haploid genome read coverage of 30-40 times using HiFi sequencing is required for phasing of the leaf rust genome, with 0.7% heterozygosity, and that HiFi sequencing resolves genomic regions with low heterozygosity that are otherwise collapsed in the Nanopore assembly. CONCLUSIONS: This first Hi-C based phasing pipeline for dikaryons and comparison of long-read sequencing technologies will inform future genome assembly and haplotype phasing projects in other non-haploid organisms.

The stem rust effector protein AvrSr50 escapes Sr50 recognition by a substitution in a single surface-exposed residue.

Pathogen effectors are crucial players during plant colonisation and infection. Plant resistance mostly relies on effector recognition to activate defence responses. Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment. Here we examined the role of genetic diversity of the stem rust (Puccinia graminis f. sp. tritici (Pgt)) AvrSr50 gene in determining recognition by the corresponding wheat Sr50 resistance gene. We solved the crystal structure of a natural variant of AvrSr50 and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence. We report that AvrSr50 can escape recognition by Sr50 through different mechanisms including DNA insertion, stop codon loss or by amino-acid variation involving a single substitution of the AvrSr50 surface-exposed residue Q121. We also report structural homology of AvrSr50 to cupin superfamily members and carbohydrate-binding modules indicating a potential role in binding sugar moieties. This study identifies key polymorphic sites present in AvrSr50 alleles from natural stem rust populations that play important roles to escape from Sr50 recognition. This constitutes an important step to better understand Pgt effector evolution and to monitor AvrSr50 variants in natural rust populations.

The stem rust fungus Puccinia graminis f. sp. tritici induces centromeric small RNAs during late infection that are associated with genome-wide DNA methylation.

BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.

Genomics accelerated isolation of a new stem rust avirulence gene-wheat resistance gene pair.

Stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt) is a devastating disease of the global staple crop wheat. Although this disease was largely controlled in the latter half of the twentieth century, new virulent strains of Pgt, such as Ug99, have recently evolved1,2. These strains have caused notable losses worldwide and their continued spread threatens global wheat production. Breeding for disease resistance provides the most cost-effective control of wheat rust diseases3. A number of rust resistance genes have been characterized in wheat and most encode immune receptors of the nucleotide-binding leucine-rich repeat (NLR) class4, which recognize pathogen effector proteins known as avirulence (Avr) proteins5. However, only two Avr genes have been identified in Pgt so far, AvrSr35 and AvrSr50 (refs. 6,7), and none in other cereal rusts8,9. The Sr27 resistance gene was first identified in a wheat line carrying an introgression of the 3R chromosome from Imperial rye10. Although not deployed widely in wheat, Sr27 is widespread in the artificial crop species Triticosecale (triticale), which is a wheat-rye hybrid and is a host for Pgt11,12. Sr27 is effective against Ug99 (ref. 13) and other recent Pgt strains14,15. Here, we identify both the Sr27 gene in wheat and the corresponding AvrSr27 gene in Pgt and show that virulence to Sr27 can arise experimentally and in the field through deletion mutations, copy number variation and expression level polymorphisms at the AvrSr27 locus.

A five-transgene cassette confers broad-spectrum resistance to a fungal rust pathogen in wheat.

Breeding wheat with durable resistance to the fungal pathogen Puccinia graminis f. sp. tritici (Pgt), a major threat to cereal production, is challenging due to the rapid evolution of pathogen virulence. Increased durability and broad-spectrum resistance can be achieved by introducing more than one resistance gene, but combining numerous unlinked genes by breeding is laborious. Here we generate polygenic Pgt resistance by introducing a transgene cassette of five resistance genes into bread wheat as a single locus and show that at least four of the five genes are functional. These wheat lines are resistant to aggressive and highly virulent Pgt isolates from around the world and show very high levels of resistance in the field. The simple monogenic inheritance of this multigene locus greatly simplifies its use in breeding. However, a new Pgt isolate with virulence to several genes at this locus suggests gene stacks will need strategic deployment to maintain their effectiveness.

Emergence of the Ug99 lineage of the wheat stem rust pathogen through somatic hybridisation.

Parasexuality contributes to diversity and adaptive evolution of haploid (monokaryotic) fungi. However, non-sexual genetic exchange mechanisms are not defined in dikaryotic fungi (containing two distinct haploid nuclei). Newly emerged strains of the wheat stem rust pathogen, Puccinia graminis f. sp. tritici (Pgt), such as Ug99, are a major threat to global food security. Here, we provide genomics-based evidence supporting that Ug99 arose by somatic hybridisation and nuclear exchange between dikaryons. Fully haplotype-resolved genome assembly and DNA proximity analysis reveal that Ug99 shares one haploid nucleus genotype with a much older African lineage of Pgt, with no recombination or chromosome reassortment. These findings indicate that nuclear exchange between dikaryotes can generate genetic diversity and facilitate the emergence of new lineages in asexual fungal populations.

De Novo Genome Assembly and Comparative Genomics of the Barley Leaf Rust Pathogen Puccinia hordei Identifies Candidates for Three Avirulence Genes.

Puccinia hordei (Ph) is a damaging pathogen of barley throughout the world. Despite its importance, almost nothing is known about the genomics of this pathogen, and a reference genome is lacking. In this study, the first reference genome was assembled for an Australian isolate of Ph ("Ph560") using long-read SMRT sequencing. A total of 838 contigs were assembled, with a total size of 207 Mbp. This included both haplotype collapsed and separated regions, consistent with an estimated haploid genome size of about 150Mbp. An annotation pipeline that combined RNA-Seq of Ph-infected host tissues and homology to proteins from four other Puccinia species predicted 25,543 gene models of which 1,450 genes were classified as encoding secreted proteins based on the prediction of a signal peptide and no transmembrane domain. Genome resequencing using short-read technology was conducted for four additional Australian strains, Ph612, Ph626, Ph608 and Ph584, which are considered to be simple mutational derivatives of Ph560 with added virulence to one or two of three barley leaf rust resistance genes (viz. Rph3, Rph13 and Rph19). To identify candidate genes for the corresponding avirulence genes AvrRph3, AvrRph13 and AvrRph19, genetic variation in predicted secreted protein genes between the strains was correlated to the virulence profiles of each, identifying 35, 29 and 46 candidates for AvrRph13, AvrRph3 and AvrRph19, respectively. The identification of these candidate genes provides a strong foundation for future efforts to isolate these three avirulence genes, investigate their biological properties, and develop new diagnostic tests for monitoring pathogen virulence.

Loss of AvrSr50 by somatic exchange in stem rust leads to virulence for Sr50 resistance in wheat.

Race-specific resistance genes protect the global wheat crop from stem rust disease caused by Puccinia graminis f. sp. tritici (Pgt) but are often overcome owing to evolution of new virulent races of the pathogen. To understand virulence evolution in Pgt, we identified the protein ligand (AvrSr50) recognized by the Sr50 resistance protein. A spontaneous mutant of Pgt virulent to Sr50 contained a 2.5 mega-base pair loss-of-heterozygosity event. A haustorial secreted protein from this region triggers Sr50-dependent defense responses in planta and interacts directly with the Sr50 protein. Virulence alleles of AvrSr50 have arisen through DNA insertion and sequence divergence, and our data provide molecular evidence that in addition to sexual recombination, somatic exchange can play a role in the emergence of new virulence traits in Pgt.

Genome analysis and avirulence gene cloning using a high-density RADseq linkage map of the flax rust fungus, Melampsora lini.

BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.

Changing the Game: Using Integrative Genomics to Probe Virulence Mechanisms of the Stem Rust Pathogen Puccinia graminis f. sp. tritici.

The recent resurgence of wheat stem rust caused by new virulent races of Puccinia graminis f. sp. tritici (Pgt) poses a threat to food security. These concerns have catalyzed an extensive global effort toward controlling this disease. Substantial research and breeding programs target the identification and introduction of new stem rust resistance (Sr) genes in cultivars for genetic protection against the disease. Such resistance genes typically encode immune receptor proteins that recognize specific components of the pathogen, known as avirulence (Avr) proteins. A significant drawback to deploying cultivars with single Sr genes is that they are often overcome by evolution of the pathogen to escape recognition through alterations in Avr genes. Thus, a key element in achieving durable rust control is the deployment of multiple effective Sr genes in combination, either through conventional breeding or transgenic approaches, to minimize the risk of resistance breakdown. In this situation, evolution of pathogen virulence would require changes in multiple Avr genes in order to bypass recognition. However, choosing the optimal Sr gene combinations to deploy is a challenge that requires detailed knowledge of the pathogen Avr genes with which they interact and the virulence phenotypes of Pgt existing in nature. Identifying specific Avr genes from Pgt will provide screening tools to enhance pathogen virulence monitoring, assess heterozygosity and propensity for mutation in pathogen populations, and confirm individual Sr gene functions in crop varieties carrying multiple effective resistance genes. Toward this goal, much progress has been made in assembling a high quality reference genome sequence for Pgt, as well as a Pan-genome encompassing variation between multiple field isolates with diverse virulence spectra. In turn this has allowed prediction of Pgt effector gene candidates based on known features of Avr genes in other plant pathogens, including the related flax rust fungus. Upregulation of gene expression in haustoria and evidence for diversifying selection are two useful parameters to identify candidate Avr genes. Recently, we have also applied machine learning approaches to agnostically predict candidate effectors. Here, we review progress in stem rust pathogenomics and approaches currently underway to identify Avr genes recognized by wheat Sr genes.

The wheat durable, multipathogen resistance gene Lr34 confers partial blast resistance in rice.

The wheat gene Lr34 confers durable and partial field resistance against the obligate biotrophic, pathogenic rust fungi and powdery mildew in adult wheat plants. The resistant Lr34 allele evolved after wheat domestication through two gain-of-function mutations in an ATP-binding cassette transporter gene. An Lr34-like fungal disease resistance with a similar broad-spectrum specificity and durability has not been described in other cereals. Here, we transformed the resistant Lr34 allele into the japonica rice cultivar Nipponbare. Transgenic rice plants expressing Lr34 showed increased resistance against multiple isolates of the hemibiotrophic pathogen Magnaporthe oryzae, the causal agent of rice blast disease. Host cell invasion during the biotrophic growth phase of rice blast was delayed in Lr34-expressing rice plants, resulting in smaller necrotic lesions on leaves. Lines with Lr34 also developed a typical, senescence-based leaf tip necrosis (LTN) phenotype. Development of LTN during early seedling growth had a negative impact on formation of axillary shoots and spikelets in some transgenic lines. One transgenic line developed LTN only at adult plant stage which was correlated with lower Lr34 expression levels at seedling stage. This line showed normal tiller formation and more importantly, disease resistance in this particular line was not compromised. Interestingly, Lr34 in rice is effective against a hemibiotrophic pathogen with a lifestyle and infection strategy that is different from obligate biotrophic rusts and mildew fungi. Lr34 might therefore be used as a source in rice breeding to improve broad-spectrum disease resistance against the most devastating fungal disease of rice.

The different effects of starch synthase IIa mutations or variation on endosperm amylose content of barley, wheat and rice are determined by the distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma.

KEY MESSAGE: The distribution of starch synthase I and starch branching enzyme IIb between the starch granule and amyloplast stroma plays an important role in determining endosperm amylose content of cereal grains. Starch synthase IIa (SSIIa) catalyses the polymerisation of intermediate length glucan chains of amylopectin in the endosperm of cereals. Mutations of SSIIa genes in barley and wheat and inactive SSIIa variant in rice induce similar effects on the starch structure and the amylose content, but the severity of the phenotypes is different. This study compared the levels of transcripts and partitioning of proteins of starch synthase I (SSI) and starch branching enzyme IIb (SBEIIb) inside and outside the starch granules in the developing endosperms of these ssIIa mutants and inactive SSIIa variant. Pleiotropic effects on starch granule-bound proteins suggested that the different effects of SSIIa mutations on endosperm amylose content of barley, wheat and rice are determined by the distribution of SSI and SBEIIb between the starch granule and amyloplast stroma in cereals. Regulation of starch synthesis in ssIIa mutants and inactive SSIIa variant may be at post-translational level or the altered amylopectin structure deprives the affinity of SSI and SBEIIb to amylopectin.

Diversifying selection in the wheat stem rust fungus acts predominantly on pathogen-associated gene families and reveals candidate effectors.

Plant pathogens cause severe losses to crop plants and threaten global food production. One striking example is the wheat stem rust fungus, Puccinia graminis f. sp. tritici, which can rapidly evolve new virulent pathotypes in response to resistant host lines. Like several other filamentous fungal and oomycete plant pathogens, its genome features expanded gene families that have been implicated in host-pathogen interactions, possibly encoding effector proteins that interact directly with target host defense proteins. Previous efforts to understand virulence largely relied on the prediction of secreted, small and cysteine-rich proteins as candidate effectors and thus delivered an overwhelming number of candidates. Here, we implement an alternative analysis strategy that uses the signal of adaptive evolution as a line of evidence for effector function, combined with comparative information and expression data. We demonstrate that in planta up-regulated genes that are rapidly evolving are found almost exclusively in pathogen-associated gene families, affirming the impact of host-pathogen co-evolution on genome structure and the adaptive diversification of specialized gene families. In particular, we predict 42 effector candidates that are conserved only across pathogens, induced during infection and rapidly evolving. One of our top candidates has recently been shown to induce genotype-specific hypersensitive cell death in wheat. This shows that comparative genomics incorporating the evolutionary signal of adaptation is powerful for predicting effector candidates for laboratory verification. Our system can be applied to a wide range of pathogens and will give insight into host-pathogen dynamics, ultimately leading to progress in strategies for disease control.

The genome sequence and effector complement of the flax rust pathogen Melampsora lini.

Rust fungi cause serious yield reductions on crops, including wheat, barley, soybean, coffee, and represent real threats to global food security. Of these fungi, the flax rust pathogen Melampsora lini has been developed most extensively over the past 80 years as a model to understand the molecular mechanisms that underpin pathogenesis. During infection, M. lini secretes virulence effectors to promote disease. The number of these effectors, their function and their degree of conservation across rust fungal species is unknown. To assess this, we sequenced and assembled de novo the genome of M. lini isolate CH5 into 21,130 scaffolds spanning 189 Mbp (scaffold N50 of 31 kbp). Global analysis of the DNA sequence revealed that repetitive elements, primarily retrotransposons, make up at least 45% of the genome. Using ab initio predictions, transcriptome data and homology searches, we identified 16,271 putative protein-coding genes. An analysis pipeline was then implemented to predict the effector complement of M. lini and compare it to that of the poplar rust, wheat stem rust and wheat stripe rust pathogens to identify conserved and species-specific effector candidates. Previous knowledge of four cloned M. lini avirulence effector proteins and two basidiomycete effectors was used to optimize parameters of the effector prediction pipeline. Markov clustering based on sequence similarity was performed to group effector candidates from all four rust pathogens. Clusters containing at least one member from M. lini were further analyzed and prioritized based on features including expression in isolated haustoria and infected leaf tissue and conservation across rust species. Herein, we describe 200 of 940 clusters that ranked highest on our priority list, representing 725 flax rust candidate effectors. Our findings on this important model rust species provide insight into how effectors of rust fungi are conserved across species and how they may act to promote infection on their hosts.

A bacterial type III secretion-based delivery system for functional assays of fungal effectors in cereals.

Large numbers of candidate effectors are being identified by genome sequencing of fungal pathogens and in planta expression studies. These effectors are both a boon and a curse for pathogens as they modulate the host cellular environment or suppress defense response to allow fungal growth as well as become targets of plant resistance (R) proteins. Recognition of a fungal effector by a plant R protein triggers a hypersensitive reaction (HR) leading to death of plant cells in and around the infection site, thus preventing further proliferation of the pathogen. Such HR induction has been used as an indicator of effector activity in functional assays of candidate effectors in dicots based on Agrobacterium-mediated transient expression. However, the Agrobacterium assay is not functional in cereal leaves. We therefore have adapted an alternative assay based on effector protein delivery using the type III secretion system (T3SS) of a non-pathogenic Pseudomonas spp. for use in wheat and other cereals. Here, we describe protocols for delivery of effector proteins into wheat and barley cells using the AvrRpm1 T3SS signal in the engineered non-pathogenic Pseudomonas fluorescens strain Effector-to-Host Analyzer (EtHAn). For ease of making expression clones we have generated the GATEWAY cloning compatible vectors. A calmodulin-dependent adenylate cyclase (Cya) reporter protein can be used as an effective marker for fusion protein delivery into wheat and barley by this system.

Genomic analysis of Xanthomonas translucens pathogenic on wheat and barley reveals cross-kingdom gene transfer events and diverse protein delivery systems.

In comparison to dicot-infecting bacteria, only limited numbers of genome sequences are available for monocot-infecting and in particular cereal-infecting bacteria. Herein we report the characterisation and genome sequence of Xanthomonas translucens isolate DAR61454 pathogenic on wheat and barley. Based on phylogenetic analysis of the ATP synthase beta subunit (atpD) gene, DAR61454 is most closely related to other X. translucens strains and the sugarcane- and banana- infecting Xanthomonas strains, but shares a type III secretion system (T3SS) with X. translucens pv. graminis and more distantly related xanthomonads. Assays with an adenylate cyclase reporter protein demonstrate that DAR61454's T3SS is functional in delivering proteins to wheat cells. X. translucens DAR61454 also encodes two type VI secretion systems with one most closely related to those found in some strains of the rice infecting strain X. oryzae pv. oryzae but not other xanthomonads. Comparative analysis of 18 different Xanthomonas isolates revealed 84 proteins unique to cereal (i.e. rice) infecting isolates and the wheat/barley infecting DAR61454. Genes encoding 60 of these proteins are found in gene clusters in the X. translucens DAR61454 genome, suggesting cereal-specific pathogenicity islands. However, none of the cereal pathogen specific proteins were homologous to known Xanthomonas spp. effectors. Comparative analysis outside of the bacterial kingdom revealed a nucleoside triphosphate pyrophosphohydrolase encoding gene in DAR61454 also present in other bacteria as well as a number of pathogenic Fusarium species, suggesting that this gene may have been transmitted horizontally from bacteria to the Fusarium lineage of pathogenic fungi. This example further highlights the importance of horizontal gene acquisition from bacteria in the evolution of fungi.

Comparative genomics of Australian isolates of the wheat stem rust pathogen Puccinia graminis f. sp. tritici reveals extensive polymorphism in candidate effector genes.

The wheat stem rust fungus Puccinia graminis f. sp. tritici (Pgt) is one of the most destructive pathogens of wheat. In this study, a draft genome was built for a founder Australian Pgt isolate of pathotype (pt.) 21-0 (collected in 1954) by next generation DNA sequencing. A combination of reference-based assembly using the genome of the previously sequenced American Pgt isolate CDL 75-36-700-3 (p7a) and de novo assembly were performed resulting in a 92 Mbp reference genome for Pgt isolate 21-0. Approximately 13 Mbp of de novo assembled sequence in this genome is not present in the p7a reference assembly. This novel sequence is not specific to 21-0 as it is also present in three other Pgt rust isolates of independent origin. The new reference genome was subsequently used to build a pan-genome based on five Australian Pgt isolates. Transcriptomes from germinated urediniospores and haustoria were separately assembled for pt. 21-0 and comparison of gene expression profiles showed differential expression in ∼10% of the genes each in germinated spores and haustoria. A total of 1,924 secreted proteins were predicted from the 21-0 transcriptome, of which 520 were classified as haustorial secreted proteins (HSPs). Comparison of 21-0 with two presumed clonal field derivatives of this lineage (collected in 1982 and 1984) that had evolved virulence on four additional resistance genes (Sr5, Sr11, Sr27, SrSatu) identified mutations in 25 HSP effector candidates. Some of these mutations could explain their novel virulence phenotypes.

A bacterial type III secretion assay for delivery of fungal effector proteins into wheat.

Large numbers of candidate effectors from fungal pathogens are being identified through whole-genome sequencing and in planta expression studies. Although Agrobacterium-mediated transient expression has enabled high-throughput functional analysis of effectors in dicot plants, this assay is not effective in cereal leaves. Here, we show that a nonpathogenic Pseudomonas fluorescens engineered to express the type III secretion system (T3SS) of P. syringae and the wheat pathogen Xanthomonas translucens can deliver fusion proteins containing T3SS signals from P. syringae (AvrRpm1) and X. campestris (AvrBs2) avirulence (Avr) proteins, respectively, into wheat leaf cells. A calmodulin-dependent adenylate cyclase reporter protein was delivered effectively into wheat and barley by both bacteria. Absence of any disease symptoms with P. fluorescens makes it more suitable than X. translucens for detecting a hypersensitive response (HR) induced by an effector protein with avirulence activity. We further modified the delivery system by removal of the myristoylation site from the AvrRpm1 fusion to prevent its localization to the plasma membrane which could inhibit recognition of an Avr protein. Delivery of the flax rust AvrM protein by the modified delivery system into transgenic tobacco leaves expressing the corresponding M resistance protein induced a strong HR, indicating that the system is capable of delivering a functional rust Avr protein. In a preliminary screen of effectors from the stem rust fungus Puccinia graminis f. sp. tritici, we identified one effector that induced a host genotype-specific HR in wheat. Thus, the modified AvrRpm1:effector-Pseudomonas fluorescens system is an effective tool for large-scale screening of pathogen effectors for recognition in wheat.