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Ralstonia pseudosolanacearum (Rps), previously known as R. solanacearum phylotypes I and III is one of the causal agents of bacterial wilt, a devastating disease that affects more than 250 plant species. Emerging Rps strains were identified infecting new hosts. P824 Rps strain was isolated from blueberry in Florida. Rps strains including PD7123 were isolated from hybrid tea roses in several countries through Europe. P781 is a representative strain of Rps commonly found on mandevilla in Florida. UW757 is a strain isolated from osteospermum plants originating in Guatemala. These strains are phylogenetically closely related and of economic importance on their respective hosts. The objective of this study is to associate the Type 3 Effectors (T3Es) repertoire of these four strains with host specificity. Candidate T3E associated with host specificity to blueberry, tea rose, osteospermum, and mandevilla were identified by sequence homology. Pathogenicity assays on 8 hosts including, blueberry, mandevilla, osteospermum and tea rose with the 4 strains showed that both P824 and PD7123 are pathogenic to blueberry and tea rose. P781 is the only strain pathogenic to mandevilla and P824 is the only strain non-pathogenic to osteospermum. Hypotheses based on correlation of T3E presence/absence and pathogenicity profiles identified 3 candidate virulence and 3 avirulence T3E for host specificity to blueberry and tea rose. Two candidate avirulence T3E were identified for mandevilla, and one candidate virulence for osteospermum. The strategy applied here can be used to reduce the number of host specificity candidate genes in closely related strains with different hosts.

The interaction between plants and pathogens is a very dynamic and complex relationship that also includes a high degree of specificity, and it is precisely this last characteristic which triggers such important responses in the survival of one or the other. The pathosystem formed by tomato (Solanum lycopersicum L.) and Fusarium oxysporum f. sp. lycopersici (Fol) has been the subject of multiple studies due to the importance of the vegetable worldwide and for the economic and ecological impact of the fungus responsible for the vascular wilt disease in tomato, causing losses that go up to 100%. One way to find alternatives for the management of any pathosystem is to know the actors involved and the mechanisms that govern the interaction through technological and scientific advances that clearly show how the interaction develops on a genetic level. This review collects the information from different scientific sources with focus on the knowledge of the fungus, tomato cultivation and plant defense applied to this pathosystem, as well as the molecular mechanisms.

The powdery mildew fungi secrete numerous Candidate Secreted Effector Proteins (CSEPs) to manipulate host immunity during infection of host plants. However, the function of most of these CSEPs in cell death suppression has not yet been established. Here, we identified several CSEPs from Blumeria graminis f. sp. hordei (Bgh) that have the potential to suppress BAX- and NtMEK2DD-triggered cell death in Nicotiana benthamiana. We further characterized two effector candidates, CSEP0139 and CSEP0182, from family six and thirty-two, respectively. CSEP0139 and CSEP0182 contain a functional signal peptide and are likely secreted effectors. Expression of either CSEP0139 or CSEP0182 suppressed cell death triggered by BAX and NtMEK2DD but not by the AVRa13/MLA13 pair in N. benthamiana. Transient overexpression of CSEP0139 or CSEP0182 also inhibited BAX-induced cell death and collapse of cytoplasm in barley cells. Furthermore, overexpression of either CSEPs significantly increased Bgh haustorial formation in barley, whereas host-induced gene silencing (HIGS) of the CSEP genes reduced haustorial formation, suggesting both CSEPs promote Bgh virulence in barley. In addition, expression of CSEP0139 and CSEP0182 reduced size of the lesions caused by the necrotrophic Botrytis cinerea in N. benthamiana. Our findings suggest that CSEP0139 and CSEP0182 may target cell death components in plants to promote fungal virulence, which extends the current understanding of the functions of Bgh CSEPs and provides an opportunity for further investigation of fungal virulence in relation to cell death pathways in host plants.

Ralstonia solanacearum causes bacterial wilt disease in many plant species. Type III-secreted effectors (T3Es) play crucial roles in bacterial pathogenesis. However, some T3Es are recognized by corresponding disease resistance proteins and activate plant immunity. In this study, we identified the R. solanacearum T3E protein RipAZ1 (Ralstonia injected protein AZ1) as an avirulence determinant in the black nightshade species Solanum americanum. Based on the S. americanum accession-specific avirulence phenotype of R. solanacearum strain Pe_26, 12 candidate avirulence T3Es were selected for further analysis. Among these candidates, only RipAZ1 induced a cell death response when transiently expressed in a bacterial wilt-resistant S. americanum accession. Furthermore, loss of ripAZ1 in the avirulent R. solanacearum strain Pe_26 resulted in acquired virulence. Our analysis of the natural sequence and functional variation of RipAZ1 demonstrated that the naturally occurring C-terminal truncation results in loss of RipAZ1-triggered cell death. We also show that the 213 amino acid central region of RipAZ1 is sufficient to induce cell death in S. americanum. Finally, we show that RipAZ1 may activate defence in host cell cytoplasm. Taken together, our data indicate that the nucleocytoplasmic T3E RipAZ1 confers R. solanacearum avirulence in S. americanum. Few avirulence genes are known in vascular bacterial phytopathogens and ripAZ1 is the first one in R. solanacearum that is recognized in black nightshades. This work thus opens the way for the identification of disease resistance genes responsible for the specific recognition of RipAZ1, which can be a source of resistance against the devastating bacterial wilt disease., This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center no. PJ01317501), the Rural Development Administration and National Research Foundation of Korea (NRF) grants funded by the Korea government (MSIT) (NRF-2019R1A2C2084705 & 2018R1A5A1023599 (SRC)), Republic of Korea. C.S. was supported by the Creative-Pioneering Researchers Program through Seoul National University. M.V.’s research was funded by the research grants (PID2019-108595RB-I00 and SEV/2015/0533) from the Spanish Government and the CERCA Program from the Catalan Government (Generalitat de Catalunya).

10 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP), The poverty of disease resistance gene reservoirs limits the breeding of crops for durable resistance against evolutionary dynamic pathogens. Zymoseptoria tritici which causes Septoria tritici blotch (STB), represents one of the most genetically diverse and devastating wheat pathogens worldwide. No fully virulent Z. tritici isolates against synthetic wheats carrying the major resistant gene Stb16q have been identified. Here, we use comparative genomics, mutagenesis and complementation to identify Stb16q, which confers broad-spectrum resistance against Z. tritici. The Stb16q gene encodes a plasma membrane cysteine-rich receptor-like kinase that was recently introduced into cultivated wheat and which considerably slows penetration and intercellular growth of the pathogen., Our works was supported by the “Fond de Soutien à l’Obtention Vegetale” (FSOV), the French National Institute for Agricultural Research (INRA), and the ETH Zurich Career Seed Grant (SEED-58 16-1). We thank Sreedhar Kilaru and Gero Steinberg for providing the strain 3D7 expressing eGFP. Confocal laser scanning microscopy experiments were performed in the Scientific Center for Optical and Electron Microscopy (ScopeM), ETH Zurich and at the SPIBOC imaging facility of the Institut Sophia Agrobiotech. B.F. was supported by INRA and the French Government (National Research Agency, ANR) through the ‘Investments for the Future’ LabEx SIGNALIFE: program reference #ANR-11-LABX-0028-01.

Wheat stem rust caused by the fungus Puccinia graminis f. sp. tritici (Pgt), is regaining prominence due to the recent emergence of virulent isolates and epidemics in Africa, Europe and Central Asia. The development and deployment of wheat cultivars with multiple stem rust resistance (Sr) genes stacked together will provide durable resistance. However, certain disease resistance genes can suppress each other or fail in particular genetic backgrounds. Therefore, the function of each Sr gene must be confirmed after incorporation into an Sr-gene stack. This is difficult when using pathogen disease assays due to epistasis from recognition of multiple avirulence (Avr) effectors. Heterologous delivery of single Avr effectors can circumvent this limitation, but this strategy is currently limited by the paucity of cloned Pgt Avrs. To accelerate Avr gene cloning, we outline a procedure to develop a mutant population of Pgt spores and select for gain-of-virulence mutants. We used ethyl methanesulphonate (EMS) to mutagenize urediniospores and create a library of > 10,000 independent mutant isolates that were combined into 16 bulks of ~658 pustules each. We sequenced random mutants and determined the average mutation density to be 1 single nucleotide variant (SNV) per 258 kb. From this, we calculated that a minimum of three independently derived gain-of-virulence mutants is required to identify a given Avr gene. We inoculated the mutant library onto plants containing Sr43, Sr44, or Sr45 and obtained 9, 4, and 14 mutants with virulence toward Sr43, Sr44, or Sr45, respectively. However, only mutants identified on Sr43 and Sr45 maintained their virulence when reinolculated onto the lines from which they were identified. We further characterized 8 mutants with virulence toward Sr43. These also maintained their virulence profile on the stem rust international differential set containing 20 Sr genes, indicating that they were most likely not accidental contaminants. In conclusion, our method allows selecting for virulent mutants toward targeted resistance (R) genes. The development of a mutant library from as little as 320 mg spores creates a resource that enables screening against several R genes without the need for multiple rounds of spore multiplication and mutagenesis.

Stripe rust is a destructive disease of wheat worldwide. Growing resistant cultivars is the most effective, easy-to-use, economical, and environmentally friendly strategy for the control of the disease. However, P. striiformis f. sp. tritici can produce new virulent races that may circumvent race-specific resistance. Therefore, understanding the genetic basis of the interactions between wheat genes for resistance and P. striiformis f. sp. tritici genes for avirulence is useful for improving cultivar resistance for more effective control of the disease. This study developed a high-quality map that facilitates genomic and genetic studies of important traits related to pathogen pathogenicity and adaptation to different environments and crop cultivars carrying different resistance genes. The information on avirulence/virulence genes identified in this study can be used for guiding breeding programs to select combinations of genes for developing new cultivars with effective resistance to mitigate this devastating disease., Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence (Avr) genes in P. striiformis f. sp. tritici, we developed a segregating population by selfing isolate 12-368 on barberry (Berberis vulgaris) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7, AvYr43, AvYr44, and AvYrExp2. Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence (Avr) genes in P. striiformis f. sp. tritici, we developed a segregating population by selfing isolate 12-368 on barberry (Berberis vulgaris) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7, AvYr43, AvYr44, and AvYrExp2. Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (

Puccinia striiformis f. sp. tritici, the causal agent of wheat stripe (yellow) rust, is an obligate, biotrophic fungus. It was difficult to study the genetics of the pathogen due to the lack of sexual reproduction. The recent discovery of alternate hosts for P. striiformis f. sp. tritici makes it possible to study inheritance and map genes involved in its interaction with plant hosts. To identify avirulence (Avr) genes in P. striiformis f. sp. tritici, we developed a segregating population by selfing isolate 12-368 on barberry (Berberis vulgaris) plants under controlled conditions. The dikaryotic sexual population segregated for avirulent/virulent phenotypes on nine Yr single-gene lines. The parental and progeny isolates were whole-genome sequenced at >30× coverage using Illumina HiSeq PE150 technology. A total of 2,637 high-quality markers were discovered by mapping the whole-genome sequencing (WGS) reads to the reference genome of strain 93-210 and used to construct a genetic map, consisting of 41 linkage groups, spanning 7,715.0 centimorgans (cM) and covering 68 Mb of the reference genome. The recombination rate was estimated to be 1.81 ± 2.32 cM/10 kb. Quantitative trait locus analysis mapped six Avr gene loci to the genetic map, including an Avr cluster harboring four Avr genes, AvYr7, AvYr43, AvYr44, and AvYrExp2 Aligning the genetic map to the reference genome identified Avr candidates and narrowed them to a small genomic region (

BackgroundThe stripe rust pathogen,Puccinia striiformisf. sp.tritici(Pst), threats world wheat production. Resistance toPstis often overcome by pathogen virulence changes, but the mechanisms of variation are not clearly understood. To determine the role of mutation inPstvirulence changes, in previous studies 30 mutant isolates were developed from a least virulent isolate using ethyl methanesulfonate (EMS) mutagenesis and phenotyped for virulence changes. The progenitor isolate was sequenced, assembled and annotated for establishing a high-quality reference genome. In the present study, the 30 mutant isolates were sequenced and compared to the wide-type isolate to determine the genomic variation and identify candidates for avirulence (Avr) genes.ResultsThe sequence reads of the 30 mutant isolates were mapped to the wild-type reference genome to identify genomic changes. After selecting EMS preferred mutations, 264,630 and 118,913 single nucleotide polymorphism (SNP) sites and 89,078 and 72,513 Indels (Insertion/deletion) were detected among the 30 mutant isolates compared to the primary scaffolds and haplotigs of the wild-type isolate, respectively. Deleterious variants including SNPs and Indels occurred in 1866 genes. Genome wide association analysis identified 754 genes associated with avirulence phenotypes. A total of 62 genes were found significantly associated to 16 avirulence genes after selection through six criteria for putative effectors and degree of association, including 48 genes encoding secreted proteins (SPs) and 14 non-SP genes but with high levels of association (P ≤ 0.001) to avirulence phenotypes. Eight of the SP genes were identified as avirulence-associated effectors with high-confidence as they met five or six criteria used to determine effectors.ConclusionsGenome sequence comparison of the mutant isolates with the progenitor isolate unraveled a large number of mutation sites along the genome and identified high-confidence effector genes as candidates for avirulence genes inPst.Since the avirulence gene candidates were identified from associated SNPs and Indels caused by artificial mutagenesis, these avirulence gene candidates are valuable resources for elucidating the mechanisms of the pathogen pathogenicity, and will be studied to determine their functions in the interactions between the wheat host and thePstpathogen.

Fifty-two infection-induced Phytophthora infestans RXLR effectors have diverse subcellular localizations, and the majority significantly enhance infection when expressed in leaves. Combining effectors targeting different defence pathways additively enhances colonization., Oomycetes such as the potato blight pathogen Phytophthora infestans deliver RXLR effectors into plant cells to manipulate host processes and promote disease. Knowledge of where they localize inside host cells is important in understanding their function. Fifty-two P. infestans RXLR effectors (PiRXLRs) up-regulated during early stages of infection were expressed as fluorescent protein (FP) fusions inside cells of the model host Nicotiana benthamiana. FP–PiRXLR fusions were predominantly nucleo-cytoplasmic, nuclear, or plasma membrane-associated. Some also localized to the endoplasmic reticulum, mitochondria, peroxisomes, or microtubules, suggesting diverse sites of subcellular activity. Seven of the 25 PiRXLRs examined during infection accumulated at sites of haustorium penetration, probably due to co-localization with host target processes; Pi16663 (Avr1), for example, localized to Sec5-associated mobile bodies which showed perihaustorial accumulation. Forty-five FP–RXLR fusions enhanced pathogen leaf colonization when expressed in Nicotiana benthamiana, revealing that their presence was beneficial to infection. Co-expression of PiRXLRs that target and suppress different immune pathways resulted in an additive enhancement of colonization, indicating the potential to study effector combinations using transient expression assays. We provide a broad platform of high confidence P. infestans effector candidates from which to investigate the mechanisms, singly and in combination, by which this pathogen causes disease.

Summary The oomycete pathogens Phytophthora infestans and P. capsici cause significant crop losses world‐wide, threatening food security. In each case, pathogenicity factors, called RXLR effectors, contribute to virulence. Some RXLRs are perceived by resistance proteins to trigger host immunity, but our understanding of the demographic processes and adaptive evolution of pathogen virulence remains poor.Here, we describe PenSeq, a highly efficient enrichment sequencing approach for genes encoding pathogenicity determinants which, as shown for the infamous potato blight pathogen Phytophthora infestans, make up, See also the Commentary on this article by Kale, 221: 1177–1179.

International audience; Fungal pathogens can secrete hundreds of effectors, some of which are known to promote host susceptibility. This biological complexity, together with the lack of genetic tools in some fungi, presents a substantial challenge to develop a broad picture of the mechanisms these pathogens use for host manipulation. Nevertheless, recent advances in understanding individual effector functions are beginning to flesh out our view of fungal pathogenesis. This review discusses some of the latest findings that illustrate how effectors from diverse species use similar strategies to modulate plant physiology to their advantage. We also summarize recent breakthroughs in the identification of effectors from challenging systems, like obligate biotrophs, and emerging concepts such as the 'iceberg model' to explain how the activation of plant immunity can be turned off by effectors with suppressive activity.

Magnaporthe oryzae infects rice, wheat, and many grass species in the Poaceae family by secreting protein effectors. Here, we analyzed the distribution, sequence variation, and genomic context of effector candidate (EFC) genes in 31 isolates that represent five pathotypes of M. oryzae, three isolates of M. grisea, a sister species of M. oryzae, and one strain each for eight species in the family Magnaporthaceae to investigate how the host range expansion of M. oryzae has likely affected the evolution of effectors. We used the EFC genes of M. oryzae strain 70-15, whose genome has served as a reference for many comparative genomics analyses, to identify their homologs in these strains. We also analyzed the previously characterized avirulence (AVR) genes and single-copy orthologous (SCO) genes in these strains, which showed that the EFC and AVR genes evolved faster than the SCO genes. The EFC and AVR repertoires among M. oryzae pathotypes varied widely probably because adaptation to individual hosts exerted different types of selection pressure. Repetitive DNA elements appeared to have caused the variation of some EFC genes. Lastly, we analyzed expression patterns of the AVR and EFC genes to test the hypothesis that such genes are preferentially expressed during host infection. This comprehensive dataset serves as a foundation for future studies on the genetic basis of the evolution and host specialization in M. oryzae.

Magnaporthe oryzae infects rice, wheat, and many grass species in the Poaceae family by secreting protein effectors. Here, we analyzed the distribution, sequence variation, and genomic context of effector candidate (EFC) genes in 31 isolates that represent five pathotypes of M. oryzae, three isolates of M. grisea, a sister species of M. oryzae, and one strain each for eight species in the family Magnaporthaceae to investigate how the host range expansion of M. oryzae has likely affected the evolution of effectors. We used the EFC genes of M. oryzae strain 70-15, whose genome has served as a reference for many comparative genomics analyses, to identify their homologs in these strains. We also analyzed the previously characterized avirulence (AVR) genes and single-copy orthologous (SCO) genes in these strains, which showed that the EFC and AVR genes evolved faster than the SCO genes. The EFC and AVR repertoires among M. oryzae pathotypes varied widely probably because adaptation to individual hosts exerted different types of selection pressure. Repetitive DNA elements appeared to have caused the variation of some EFC genes. Lastly, we analyzed expression patterns of the AVR and EFC genes to test the hypothesis that such genes are preferentially expressed during host infection. This comprehensive dataset serves as a foundation for future studies on the genetic basis of the evolution and host specialization in M. oryzae.

© 2019 The Authors. Molecular Plant Pathology Published by British Society for Plant Pathology and John Wiley & Sons Ltd Bacterial fruit blotch (BFB) caused by Acidovorax citrulli is one of the most important bacterial diseases of cucurbits worldwide. However, the mechanisms associated with A. citrulli pathogenicity and genetics of host resistance have not been extensively investigated. We idenitfied Nicotiana benthamiana and Nicotiana tabacum as surrogate hosts for studying A. citrulli pathogenicity and non-host resistance triggered by type III secreted (T3S) effectors. Two A. citrulli strains, M6 and AAC00-1, that represent the two major groups amongst A. citrulli populations, induced disease symptoms on N. benthamiana, but triggered a hypersensitive response (HR) on N. tabacum plants. Transient expression of 19 T3S effectors from A. citrulli in N. benthamiana leaves revealed that three effectors, Aave_1548, Aave_2708, and Aave_2166, trigger water-soaking-like cell death in N. benthamiana. Aave_1548 knockout mutants of M6 and AAC00-1 displayed reduced virulence on N. benthamiana and melon (Cucumis melo L.). Transient expression of Aave_1548 and Aave_2166 effectors triggered a non-host HR in N. tabacum, which was dependent on the functionality of the immune signalling component, NtSGT1. Hence, employing Nicotiana species as surrogate hosts for studying A. citrulli pathogenicity may help characterize the function of A. citrulli T3S effectors and facilitate the development of new strategies for BFB management. 2nd (Edition) Published (Publication status)

Pathogens and plants are in a constant battle with one another, the result of which is either the restriction of pathogen growth via constitutive or induced plant defense responses or the pathogen colonization of plant cells and tissues that cause disease. Elicitins are a group of highly conserved proteins produced by certain oomycete species, and their sterol binding ability is recognized as an important feature in sterol–auxotrophic oomycetes. Elicitins also orchestrate other aspects of the interactions of oomycetes with their plant hosts. The function of elicitins as avirulence or virulence factors is controversial and is dependent on the host species, and despite several decades of research, the function of these proteins remains elusive. We summarize here our current understanding of elicitins as either defense-promoting or defense-suppressing agents and propose that more recent approaches such as the use of ‘omics’ and gene editing can be used to unravel the role of elicitins in host–pathogen interactions. A better understanding of the role of elicitins is required and deciphering their role in host–pathogen interactions will expand the strategies that can be adopted to improve disease resistance and reduce crop losses.

International audience; Leptosphaeria maculans, the causal agent of blackleg disease, interacts with Brassica napus (oilseed rape, canola) and other Brassica hosts in a gene-for-gene manner. The avirulence gene AvrLmJ1 has been cloned previously and shown to interact with an unidentified Brassica juncea resistance gene. In this study, we show that the AvrLmJ1 gene maps to the same position as the AvrLm5 locus. Furthermore, isolates complemented with the AvrLmJ1 locus confer avirulence towards B. juncea genotypes harbouring Rlm5. These findings demonstrate that AvrLmJ1 is AvrLm5 and highlight the need for shared resources to characterize accurately avirulence and/or resistance genes.

[EN] Tomato spotted wilt virus (TSWV) is worldwide spread and causes production losses in many important horticultural crops such as tomato and pepper. Breeding resistant cultivars has been the most successful method for disease control. So far, only two resistance genes have been found to confer resistance against a wide spectrum of TSWV isolates: Sw-5 in tomato and Tsw in pepper. However, TSWV resistance-breaking isolates have emerged in different countries after a few years of implementation of resistant cultivars. In this paper we report the first complete nucleotide sequence of three TSWV isolates from Spain with different biotypes according to the ability of overcoming resistance: LL-N.05 (wild type, WT), Pujol1TL3 (Sw-5 resistance breaking, SBR) and PVR (Tsw resistance-breaking, TBR). The genome of these TSWV isolates consisted of three genomic segments: L (8913-8914 nt), M (4752-4825 nt) and S (2924-2961 nt). Variations in nucleotide sequences and genomic lengths among the different virus biotypes are reported here. Phylogenetic analysis of the five TSWV open reading frames showed evidences of reassortment between genomic segments of LL-N.05 and Pujol1TL3, which was supported by analysis with different recombination-detecting algorithms. Genetic distances were uncorrelated to biotype, host or geographic location., D.E.D. was the recipient of a predoctoral FPU fellowship from the Spanish Ministry of Education, Culture and Sports. This work was funded in part by grants RTA2008-00010-C03 and RTA2013-00047-C02 from Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria (INIA) and grants ACOMP/2009/103, ACOMP/2010/085 and ACOMP/2011/078 from Generalitat Valenciana. Work in the J.A.D. laboratory was supported by grant BIO2011-26741 from the Spanish Ministerio de Economia y Competitividad. We thank Veronica Aragones (IBMCP, CSIC-UPV) for technical assistance.

Bacterial phytopathogen type III secreted (T3S) effectors have been strongly implicated in altering the interaction of pathogens with host plants. Therefore, it is useful to characterize the whole effector repertoire of a pathogen to understand the interplay of effectors in plants. Pseudomonas syringae pv. actinidiae is a causal agent of kiwifruit canker disease. In this study, we generated an Agrobacterium-mediated transient expression library of YFP-tagged T3S effectors from two strains of Psa, Psa-NZ V13 and Psa-NZ LV5, in order to gain insight into their mode of action in Nicotiana tabacum and N. benthamiana. Determining the subcellular localization of effectors gives an indication of the possible host targets of effectors. A confocal microscopy assay detecting YFP-tagged Psa effectors revealed that the nucleus, cytoplasm and cell periphery are major targets of Psa effectors. Agrobacterium-mediated transient expression of multiple Psa effectors induced HR-like cell death (HCD) in Nicotiana spp., suggesting that multiple Psa effectors may be recognized by Nicotiana spp.. Virus-induced gene silencing (VIGS) of several known plant immune regulators, EDS1, NDR1, or SGT1 specified the requirement of SGT1 in HCD induced by several Psa effectors in N. benthamiana. In addition, the suppression activity of Psa effectors on HCD-inducing proteins and PTI was assessed. Psa effectors showed differential suppression activities on each HCD inducer or PTI. Taken together, our Psa effector repertoire analysis highlights the great diversity of T3S effector functions in planta.

Summary Candidate effectors from lettuce downy mildew (Bremia lactucae) enable high‐throughput germplasm screening for the presence of resistance (R) genes. The nonhost species Lactuca saligna comprises a source of B. lactucae R genes that has hardly been exploited in lettuce breeding. Its cross‐compatibility with the host species L. sativa enables the study of inheritance of nonhost resistance (NHR).We performed transient expression of candidate RXLR effector genes from B. lactucae in a diverse Lactuca germplasm set. Responses to two candidate effectors (BLR31 and BLN08) were genetically mapped and tested for co‐segregation with disease resistance.BLN08 induced a hypersensitive response (HR) in 55% of the L. saligna accessions, but responsiveness did not co‐segregate with resistance to Bl:24. BLR31 triggered an HR in 5% of the L. saligna accessions, and revealed a novel R gene providing complete B. lactucae race Bl:24 resistance. Resistant hybrid plants that were BLR31 nonresponsive indicated other unlinked R genes and/or nonhost QTLs.We have identified a candidate avirulence effector of B. lactucae (BLR31) and its cognate R gene in L. saligna. Concurrently, our results suggest that R genes are not required for NHR of L. saligna.

Phytophthora sojae is an oomycete responsible for seed, root and stem rot of soybean plants. Managing this disease relies on growing soybean cultivars with race-specific resistance (Rps) genes that deliver complete host immunity in the presence of corresponding pathogen avirulence (Avr) effector proteins. The aims of this study were to characterize virulence towards Rps2 among different P. sojae strains, track the inheritance of this trait, and attempt to identify an Avr2 gene. Fifteen P. sojae strains were tested for virulence towards Rps2 and crosses were performed between selected virulent and avirulent strains to follow the inheritance of virulence. Although parental strains were consistent in their virulence phenotype, many progeny were unstable. Of two candidate genes for Avr2 tested, neither showed co-segregation with the Rps2-virulence trait. Overall, results indicate that virulence towards Rps2 is inherited in a non-Mendelian fashion; the factors responsible could include epistasis, gene conversion, or other epigenetic phenomena.

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 towards 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 simultaneous 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. Towards 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. Up regulation 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

Most fungal effectors characterized so far are species-specific and facilitate virulence on a particular host plant. During infection of its host tomato, Cladosporium fulvum secretes effectors that function as virulence factors in the absence of cognate Cf resistance proteins and induce effector-triggered immunity in their presence. Here we show that homologs of the C. fulvum Avr4 and Ecp2 effectors are present in other pathogenic fungi of the Dothideomycete class, including Mycosphaerella fijiensis , the causal agent of black Sigatoka disease of banana. We demonstrate that the Avr4 homolog of M. fijiensis is a functional ortholog of C. fulvum Avr4 that protects fungal cell walls against hydrolysis by plant chitinases through binding to chitin and, despite the low overall sequence homology, triggers a Cf-4-mediated hypersensitive response (HR) in tomato. Furthermore, three homologs of C. fulvum Ecp2 are found in M. fijiensis , one of which induces different levels of necrosis or HR in tomato lines that lack or contain a putative cognate Cf-Ecp2 protein, respectively. In contrast to Avr4, which acts as a defensive virulence factor, M. fijiensis Ecp2 likely promotes virulence by interacting with a putative host target causing host cell necrosis, whereas Cf-Ecp2 could possibly guard the virulence target of Ecp2 and trigger a Cf-Ecp2-mediated HR. Overall our data suggest that Avr4 and Ecp2 represent core effectors that are collectively recognized by single cognate Cf-proteins. Transfer of these Cf genes to plant species that are attacked by fungi containing these cognate core effectors provides unique ways for breeding disease-resistant crops.

XopJ is a Xanthomonas type III effector protein that promotes bacterial virulence on susceptible pepper plants through the inhibition of the host cell proteasome and a resultant suppression of salicylic acid (SA) - dependent defense responses. We show here that Nicotiana benthamiana leaves transiently expressing XopJ display hypersensitive response (HR) -like symptoms when exogenously treated with SA. This apparent avirulence function of XopJ was further dependent on effector myristoylation as well as on an intact catalytic triad, suggesting a requirement of its enzymatic activity for HR-like symptom elicitation. The ability of XopJ to cause a HR-like symptom development upon SA treatment was lost upon silencing of SGT1 and NDR1, respectively, but was independent of EDS1 silencing, suggesting that XopJ is recognized by an R protein of the CC-NBS-LRR class. Furthermore, silencing of NPR1 abolished the elicitation of HR-like symptoms in XopJ expressing leaves after SA application. Measurement of the proteasome activity indicated that proteasome inhibition by XopJ was alleviated in the presence of SA, an effect that was not observed in NPR1 silenced plants. Our results suggest that XopJ - triggered HR-like symptoms are closely related to the virulence function of the effector and that XopJ follows a two-signal model in order to elicit a response in the non-host plant N. benthamiana.

International audience; Leptosphaeria maculans is a hemibiotophic ascomycete which causes stem canker of oilseed rape. That phytopathogenic fungus interacts with its host (Brassica napus) according to the gene-for-gene concept. The most economically and environment friendly method of control of stem canker is the genetic control by using host resistance. Single gene resistance is extremely efficient, but races of the pathogen virulent towards a resistance gene can appear in a few years and necessitates continuously new breeding programs. Moreover, specific resistances are rare in oilseed rape, and a lot of efforts are made to find other resistance genes in other Brassica species. To date, 11 interactions were genetically characterized between L. maculans avirulence genes and corresponding resistance genes in Brassica, and 5 of those avirulence genes were cloned. Recently, the avirulence gene AvrLm10 which is recognized by the resistance gene Rlm10 of the black mustard (Brassica nigra) has been cloned. AvrLm10 corresponds in fact to two avirulence genes AvrLm10_1 and AvrLm10_2 which are located in the same AT-rich genomic region. They encore for small secreted proteins (SSP), are co-regulated and over-expressed 7 days post-infection. Each of them is necessary but not sufficient to induce resistance towards Rlm10. Silencing of one of those genes is sufficient to abolish recognition by Rlm10. Silencing by RNA interference of AvrLm10-1 induces an increase of lesion size on oilseed rape leaves while silencing of AvrLm10-2 has no major effect on aggressiveness of the fungus. That interaction of two avirulence genes against one resistance gene is therefore different from the classical gene-for-gene concept. It suggests that AvrLm10_1 and AvrLm10_2 could directly interact and / or that they could target the same plant protein. A Y2H screen suggested a direct interaction between AvrLm10-1 and AvrLm10-2. This interaction was confirmed with Bimolecular Fluorescence Complementation (BiFC) experiments. Coimmunoprecipitation experiments are also in progress to confirm this interaction.

présentation sans résumé; Deciphering the role of effectors during plant infection and chromatin-based control of their expression in the plant pathogenic fungus Leptosphaeria maculans. Séminaire à l'Université de Kiel

présentation sans résumé; Deciphering the role of effectors during plant infection and the chromatin-based control of their expression in the plant pathogenic fungus Leptosphaeria maculans. Séminaire à l'Université de Maynooth

Pensec, F., Lebeau, A., Daunay, M. C., Chiroleu, F., Guidot, A., and Wicker, E. 2015. Towards the identification of type III effectors associated with Ralstonia solanacearum virulence on tomato and eggplant. Phytopathology 105:1529-1544. For the development of pathogen-informed breeding strategies, identifying the microbial genes involved in interactions with the plant is a critical step. To identify type III effector (T3E) repertoires associated with virulence of the bacterial wilt pathogen Ralstonia solanacearum on Solanaceous crops, we used an original association genetics approach combining DNA microarray data and pathogenicity data on resistant eggplant, pepper, and tomato accessions. From this first screen, 25 T3Es were further full-length polymerase chain reaction-amplified within a 35-strain field collection, to assess their distribution and allelic diversity. Six T3E repertoire groups were identified, within which 11 representative strains were chosen to challenge the bacterial wilt-resistant eggplants 'Dingras multiple Purple' and 'AG91-25', and tomato Hawaii7996. The virulence or avirulence phenotypes could not be explained by specific T3E repertoires, but rather by individual T3E genes. We identified seven highly avirulence-associated genes, among which ripP2, primarily referenced as conferring avirulence to Arabidopsis thaliana. Interestingly, no T3E was associated with avirulence to both eggplants. Highly virulence-associated genes were also identified: ripA5_2, ripU, and ripV2. This study should be regarded as a first step toward investigating both avirulence and virulence function of the highlighted genes, but also their evolutionary dynamics in natural R. solanacearum populations.

A limited number of fungi can cause wilting disease in plants through colonization of the vascular system, the most well-known being Verticillium dahliae and Fusarium oxysporum. Like all pathogenic microorganisms, vascular wilt fungi secrete proteins during host colonization. Whole-genome sequencing and proteomics screens have identified many of these proteins, including small, usually cysteine-rich proteins, necrosis-inducing proteins and enzymes. Gene deletion experiments have provided evidence that some of these proteins are required for pathogenicity, while the role of other secreted proteins remains enigmatic. On the other hand, the plant immune system can recognize some secreted proteins or their actions, resulting in disease resistance. We give an overview of proteins currently known to be secreted by vascular wilt fungi and discuss their role in pathogenicity and plant immunity.

Plant-invading microbes betray their presence to a plant by exposure of antigenic molecules such as small, secreted proteins called 'effectors'. In Fusarium oxysporum f. sp. lycopersici (Fol) we identified a pair of effector gene candidates, AVR2-SIX5, whose expression is controlled by a shared promoter. The pathogenicity of AVR2 and SIX5 Fol knockouts was assessed on susceptible and resistant tomato (Solanum lycopersicum) plants carrying I-2. The I-2 NB-LRR protein confers resistance to Fol races carrying AVR2. Like Avr2, Six5 was found to be required for full virulence on susceptible plants. Unexpectedly, each knockout could breach I-2-mediated disease resistance. So whereas Avr2 is sufficient to induce I-2-mediated cell death, Avr2 and Six5 are both required for resistance. Avr2 and Six5 interact in yeast two-hybrid assays as well as in planta. Six5 and Avr2 accumulate in xylem sap of plants infected with the reciprocal knockouts, showing that lack of I-2 activation is not due to a lack of Avr2 accumulation in the SIX5 mutant. The effector repertoire of a pathogen determines its host specificity and its ability to manipulate plant immunity. Our findings challenge an oversimplified interpretation of the gene-for-gene model by showing requirement of two fungal genes for immunity conferred by one resistance gene.

The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound. Government of Egypt Scholarship The School of Chemistry at the University of Bristol and the Mark Evans Scholarship Kano State Government Nigeria MacArthur Foundation Bayero University Nigerian Petroleum Technology Fund Malaysian Govenment Scholarship EP/F066104/1

Summary Leptosphaeria maculans, a Dothideomycete causing stem canker on oilseed rape (Brassica napus), develops gene-for-gene interactions with its host plants. To date, nine resistance genes (Rlm1–9) have been identified in Brassica spp. The corresponding nine avirulence genes (AvrLm1–9) in L. maculans have been mapped at four independent loci, thereby revealing two clusters of three and four linked avirulence genes. Here, we report the completion of map-based cloning of AvrLm1. AvrLm1 was genetically delineated within a 7.3 centimorgan interval corresponding to a 439 kb BAC contig. AvrLm1 is a single copy gene isolated within a 269 kb non-coding, heterochromatin-like region. The region comprised a number of degenerated, nested copies of four long-terminal repeat (LTR) retrotransposons, including Pholy and three novel Gypsy-like retrotransposons. AvrLm1 restored the avirulent phenotype on Rlm1 cultivars following functional complementation of virulent isolates. AvrLm1 homologues were not detected in other Leptosphaeria species or in known fungal genomes including the closely related species Stagonospora nodorum. The predicted AvrLm1 protein is composed of 205 amino acids, of which only one is a cysteine residue. It contains a peptide signal suggesting extracellular localization. Unlike most other fungal avirulence genes, AvrLm1 is constitutively expressed, with a probable increased level of expression upon plant infection, suggesting the absence of tight regulation of AvrLm1 expression.

Comparative analysis of two Phytophthora genomes revealed overall colinearity in four genomic regions consisting of a 1.5-Mb sequence of Phytophthora sojae and a 0.9-Mb sequence of P. ramorum. In these regions with conserved synteny, the gene order is largely similar; however, genome rearrangements also have occurred. Deletions and duplications often were found in association with genes encoding secreted proteins, including effectors that are important for interaction with host plants. Among secreted protein genes, different evolutionary patterns were found. Elicitin genes that code for a complex family of highly conserved Phytophthora-specific elicitors show conservation in gene number and order, and often are clustered. In contrast, the race-specific elicitor gene Avr1b-1 appeared to be missing from the region with conserved synteny, as were its five homologs that are scattered over the four genomic regions. Some gene families encoding secreted proteins were found to be expanded in one species compared with the other. This could be the result of either repeated gene duplications in one species or specific deletions in the other. These different evolutionary patterns may shed light on the functions of these secreted proteins in the biology and pathology of the two Phytophthora spp.

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.

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.

BGPI : équipe 5; International audience; Magnaporthe oryzae, the rice blast pathogen, causes significant annual yield loss of rice worldwide. Currently, the most effective disease control approach is deployment of host resistance through introduction of resistance (R ) genes into elite cultivars. The function of each R gene relies on the specific recognition of an avirulence (AVR) gene of the pathogen. However, the introduced resistance can be broken down within a few years, due to mutation of the AVR genes in the field populations. It is known from a few case studies that AVR mutation patterns are different from one to another. Therefore, knowledge of AVR genes sequence diversity serves as fundamental background in introducing new resistance to control rice blast. In this study, we focused on a newly identified AVR gene AvrPiz-t. We PCR amplified open reading frames (ORFs) of AvrPiz-t as well as promoter regions to detect size variation at this locus in 711 isolates of M. oryzae collected from 38 countries and regions. Through sequencing and Southern hybridization of the amplified locus, strains with polymorphisms in the ORF were classified into groups based on mutation type and site. Natural selection intensity on this gene was calculated and pathogenicity assays were applied to evaluate the association between AvrPiz-t ORF/promoter polymorphism and virulence. In conclusion, sequences at the AvrPiz-t locus were revealed to contain variations at both promoter and ORF regions. This locus is undergoing a relatively strong positive selection. The diversity in coding sequence and the insertions of transposable elements in the promoter region enable M. oryzaeto evade recognition by the cognate Piz-t R gene in the host.

In the past, numerous Lycopersicon accessions have been described that harbor resistance genes to Cladosporium fulvum (Cf genes). Several Cf genes have been isolated, like Cf-4, Cf-4A and Cf-9, which are present on the short arm of Chromosome 1, and Cf-2 and Cf-5, which reside on Chromosome 6. To identify Cf genes linked to the Hcr9 cluster ”Milky Way” on the short arm of Chromosome 1, we test-crossed 66 resistant Lycopersicon accessions to the near-isogenic line Moneymaker-Cf4, and the F1s were crossed to the susceptible tomato cultivar Moneymaker. Putative linkage between an unknown Cf gene and Cf-4 was concluded based on small-scale allelic tests from an under-representation of susceptible genotypes in the progenies of 24 plants after inoculation with race 0 of C. fulvum. In this way, of the 21 resistant lines tested, 10 harbored a Cf gene that was linked to the Hcr9 Milky Way cluster. Moreover, one of the lines harboring a Cf gene closely linked to Cf-4 specifically recognizes the extracellular protein ECP5 of C. fulvum and was designated Cf-ECP5. Using a testcross population of 338 plants, we mapped Cf-ECP5 more accurately at 4 cM proximal to the Hcr9 Milky Way locus. This report shows that the method of small-scale allelic tests provides a useful tool to rapidly screen for Cf genes on the short arm of Chromosome 1. Further analysis of these Cf genes will elucidate the complex genetic organization of Cf genes on Chromosome 1 of tomato.

Upon infection of plants by pathogens, at least at the early stages of infection, the interaction between the two organisms occurs in the apoplast. To study the molecular basis of host susceptibility vs. resistance on the one hand, and pathogen virulence vs. avirulence on the other, the identification of extracellular compounds such as pathogen effectors that determine the outcome of the interaction is essential. Here, I describe the vacuum infiltration-centrifugation technique, which is an extremely simple and straightforward method to explore one of the most important battlefields of a plant–pathogen interaction; the apoplast.

African strains of Xanthomonas oryzae pv. oryzae contain fewer TAL effectors than Asian strains, and their contribution to pathogenicity is unknown. Systematic mutagenesis of tal genes was used to decipher the contribution of each of the eight TAL effector paralogs to pathogenicity of African X. oryzae pv. oryzae BAI3. A strain mutated in talC was severely affected in the production of disease symptoms. Analysis of growth in planta upon leaf-clip inoculation showed that mutant bacteria multiplied only at the site of inoculation at the apex of the leaf, suggesting a requirement for talC during colonization of vascular tissues. Such tissue-specific effect of a tal mutant is a novel phenotype, which has not yet been characterized in other xanthomonads. Microarray experiments comparing the host response of rice leaves challenged with BAI3R vs. BAI3RΔtalC were performed to identify genes targeted by TalC. A total of 120 upregulated and 21 downregulated genes were identified, among them Os11N3, which is a member of the MtN3/saliva family. Based on semiquantitative reverse transcription-polymerase chain reaction and β-glucuronidase reporter assays, we show that Os11N3 is directly upregulated by TalC and identify a TalC DNA target box within the Os11N3 upstream sequence.

Potato (Solanum tuberosum) is the world's third-largest food crop. It severely suffers from late blight, a devastating disease caused by Phytophthora infestans. This oomycete pathogen secretes host-translocated RXLR effectors that include avirulence (AVR) proteins, which are targeted by resistance (R) proteins from wild Solanum species. Most Solanum R genes appear to have coevolved with P. infestans at its center of origin in central Mexico. Various R and Avr genes were recently cloned, and here we catalog characterized R-AVR pairs. We describe the mechanisms that P. infestans employs for evading R protein recognition and discuss partial resistance and partial virulence phenotypes in the context of our knowledge of effector diversity and activity. Genome-wide catalogs of P. infestans effectors are available, enabling effectoromics approaches that accelerate R gene cloning and specificity profiling. Engineering R genes with expanded pathogen recognition has also become possible. Importantly, monitoring effector allelic diversity in pathogen populations can assist in R gene deployment in agriculture. Fil: Vleeshouwers, Vivianne G. A. A.. Wageningen UR Plant Breeding; Países Bajos Fil: Raffaele, Sylvain. Norwich Research Park; Reino Unido Fil: Vossen, Jack H.. Wageningen UR Plant Breeding; Países Bajos Fil: Champouret, Nicolas. Wageningen UR Plant Breeding; Países Bajos Fil: Oliva, Ricardo. Norwich Research Park; Reino Unido Fil: Segretin, Maria Eugenia. Norwich Research Park; Reino Unido. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Rietman, Hendrik. Wageningen UR Plant Breeding; Países Bajos Fil: Cano, Liliana M.. Norwich Research Park; Reino Unido Fil: Lokossou, Anoma. Wageningen UR Plant Breeding; Países Bajos Fil: Kessel, Geert. Plant Research International; Reino Unido Fil: Pel, Mathieu A.. Wageningen UR Plant Breeding; Países Bajos Fil: Kamoun, Sophien. Norwich Research Park; Reino Unido

International audience; In gene-for-gene systems, resistance of a plant to a pathogen is due to the ‘recognition’ by plant surveillance system (i.e. plant resistance gene product) of a pathogen avirulence determinant encoded by an avirulence gene to eventually result in triggering of plant immunity. Avirulence proteins (or products) actually are effectors involved in pathogenicity. In viruses, virtually all the proteins can behave as avirulence determinants. In all other cases,avirulence genes are extreme lydiverse,being often species or isolate/strain-specific and rarely have matches in sequence databases. They often are specifically expressed or strongly over-expressed during plant–pathogen interaction and the encoded proteins show the presence of secretion signals and translocation signals (e.g. the Type III secretion signal for bacteria). Avirulence genes seem to be submitted to high-speed diversifying selection allowing the pathogen to diversify its effector repertoire and rapidly escape recognition by the plant resistance gene.

Equipe 5; International audience; The avirulence characteristic of Magnaporthe grisea isolate TH16 corresponding to Jao Hom Nin (JHN) rice cultivar was studied by mapping population of 140 random ascospore progenies derived from the cross between B1-2 and TH16 isolates. Segregation analyses of the avirulence characteristic performing on JHN rice at the seedling and flowering stages were performed in this mapping population. We used the reference map of Guy11/2539 to choose microsatellite DNA markers for mapping the avirulence gene. The genetic map of this population was constructed from 39-microsatellite markers. The genetic map was spanned by covering seven chromosomes with an average distance of 11.9 cM per marker. In mapping population the distribution of pathogenic and non-pathogenic progenies on JHN rice were found to be fitted to 1 : 1 ratio for two of the rice stages, seedling and flowering stages. The Quantitative Trait Loci (QTL) analysis for avirulence genes corresponding to two rice stages were located at the same region on chromosome 2 between markers Pyms305 and Pyms435. The LOD score and percentage of phenotypic variance explained (PVE) on two rice stages were 5.01/16.69 and 6.73/20.26, respectively. These loci were designated as Avr-JHN(lb) and Avr-JHN(pb) corresponding to leaf and panicle blast characteristics. The findings of this study can be the initial step for positional cloning and identifying any function of avirulence genes corresponding to leaf and panicle blast characteristics.

Introduction of resistance genes from wild Solanum species into potato cultivars is considered the most promising and environmentally safe approach to achieve late blight resistance. An R-gene stacking breeding program using cisgenesis is planning to trial its products in China. To adapt this approach to local conditions, we propose to assess the allelic diversity of known avirulent genes of P. infestans from the intended introduction regions of the GM-potatoes in China. So far, we have a large (~100 isolates) and geographically diverse collection of P. infestans. We measured diversity in the isolates based on sequences and functions of several known Avr genes. The work relied on molecular and modern computer tools to examine the dynamics of the pathogen population structure and evolutionary relationships among different genotypes. We aim to give an ecological assessment of the Chinese population of P. infestans, which would be a guideline for the release of cisgenetic plants with stacked R genes

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER–containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

Magnaporthe grisea is responsible for a devastating fungal disease of rice called blast. Current control of this disease relies on resistant rice cultivars that recognize M. grisea signals corresponding to specific secreted proteins encoded by avirulence genes. The M. grisea ACE1 avirulence gene differs from others, since it controls the biosynthesis of a secondary metabolite likely recognized by rice cultivars carrying the Pi33 resistance gene. Using a transcriptional fusion between ACE1 promoter and eGFP , we showed that ACE1 is only expressed in appressoria during fungal penetration into rice and barley leaves, onion skin, and cellophane membranes. ACE1 is almost not expressed in appressoria differentiated on Teflon and Mylar artificial membranes. ACE1 expression is not induced by cellophane and plant cell wall components, demonstrating that it does not require typical host plant compounds. Cyclic AMP (cAMP) signaling mutants Δ cpkA and Δ mac1 sum1 - 99 and tetraspanin mutant Δ pls1 :: hph differentiate melanized appressoria with normal turgor but are unable to penetrate host plant leaves. ACE1 is normally expressed in these mutants, suggesting that it does not require cAMP signaling or a successful penetration event. ACE1 is not expressed in appressoria of the buf1 :: hph mutant defective for melanin biosynthesis and appressorial turgor. The addition of hyperosmotic solutes to buf1 :: hph appressoria restores appressorial development and ACE1 expression. Treatments of young wild-type appressoria with actin and tubulin inhibitors reduce both fungal penetration and ACE1 expression. These experiments suggest that ACE1 appressorium-specific expression does not depend on host plant signals but is connected to the onset of appressorium-mediated penetration.

Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell. Haustoria of the rust fungus pathogen Uromyces fabae deliver RTP1 (Rust Transferred Protein1) into host plant cells. In this work, different heterologous expression systems were used to study RTP1 biological function as well as RTP1 transfer mechanism. The first part of this thesis focused on the identification of the subcellular target compartment of RTP1 in plant cells. In this respect we could identify a functional bipartite nuclear localization signal within RTP1. However, stable and transient expression studies of RTP1 in different plant species, including the host plant Vicia faba, interfered with plant cell vitality but did not result in detection of RTP1 protein. These findings led us to propose that RTP1 interferes with plant gene expression. However, the molecular basis of this interference remains unclear. By deletion studies, we could localize the active region of RTP1 within a 45 amino acid central domain. In the second part of this study, two different lines of approaches were taken to study RTP1 transfer mechanism. First, transient expression of secreted RTP1 (sRTP1) also interfered with plant cell vitality. Addition of an endoplasmic reticulum retention signal abolished sRTP1 interference with plant cell vitality, suggesting that RTP1 can reenter the plant cell from the apoplast after secretion in the absence of the pathogen. We have identified a PEST-like region within RTP1, however, contribution of this region to the stability of RTP1 is not clear. Site directed mutagenesis analysis showed that the PEST-like region is likely to play a role during the transfer of RTP1 through plant plasma membrane. In the second line of approach, we established a recombinant delivery model, using Ustilago maydis/Zea mays pathosystem, to pursue RTP1 translocation into the plant cell. Our results indicate that U. maydis is capable of secreting high amounts of recombinant RTP1, showing similar glycosylation pattern as RTP1 secreted from rust haustoria. Our data propose the use of this model system to study RTP1 domains mediating its entry into the plant cell.

Draft genome sequences have been determined for the soybean pathogen Phytophthora sojae and the sudden oak death pathogen Phytophthora ramorum . Oömycetes such as these Phytophthora species share the kingdom Stramenopila with photosynthetic algae such as diatoms, and the presence of many Phytophthora genes of probable phototroph origin supports a photosynthetic ancestry for the stramenopiles. Comparison of the two species' genomes reveals a rapid expansion and diversification of many protein families associated with plant infection such as hydrolases, ABC transporters, protein toxins, proteinase inhibitors, and, in particular, a superfamily of 700 proteins with similarity to known oömycete avirulence genes.

▪ Abstract Over the past 20 years, the interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato has developed into a versatile experimental system for molecular plant pathology and resistance breeding. This interaction provided the resources for cloning of fungal avirulence genes for the first time and interesting clues on recognition of their extracellular products by tomato, as well as mechanisms employed by the fungus to circumvent this recognition. A wealth of information has become available on the structure and genomic organization of Cf resistance genes. The occurrence of many clustered Cf homologues allows the generation of new genes with additional recognitional specificities by reshuffling. It is anticipated that potentially all proteins secreted by C. fulvum are recognized by one or more individuals in a population of tomato genotypes, a hypothesis that has been experimentally confirmed. The future challenge will be to elucidate the mechanisms of perception of avirulence factors and the subsequent signaling eventually leading to activation of host defense responses.

Mutagenesis and sequence analyses of rhizobial genomes have revealed the presence of genes encoding type III secretion systems. Considered as a machine used by plant and animal pathogens to deliver virulence factors into their hosts, this secretion apparatus has recently been proven to play a role in symbiotic bacteria–leguminous plant interactions.