Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and... more Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolat...
Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds... more Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5-10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families.
Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and... more Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolates and host-specific lineages, the genome composition in terms of frequencies of genes putatively involved in pathogenicity (effectors, secondary metabolism, cazome) was conserved. However, 529 shared families were found only in nonrice lineages, whereas the rice lineage possessed 86 specific families absent from the nonrice genomes. Our results confirmed that the host specificity of M. oryzae isolates was associated with a divergence between lineages without major gene flow and that, despite the strong conservation of gene families between lineages, adaptation to different hosts, especially to rice, was associated with the presence of a small number of specific gene families. All information was gathered in a public database (http://genome.jouy.inra.fr/gemo).
Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellu... more Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellular nucleotide-binding, leucine-rich repeat receptors (NLR). Plant NLRs contain an N-terminal signaling domain that is mostly represented by either a Toll-interleukin1 receptor (TIR) domain or a coiled coil (CC) domain. In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. However, many paired NLRs have now been identified where both proteins are required to confer resistance to pathogens. Recent detailed studies on the Arabidopsis thaliana TIR-NLR pair RRS1 and RPS4 and on the rice CC-NLR pair RGA4 and RGA5 have revealed for the first time how such protein pairs function together. In both cases, the paired partners interact physically to form a hetero-complex receptor in which each partner plays distinct roles in effector recognition or signaling activation, highlighting a conserved mode of action of NLR pairs across both monocotyledon...
Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain prote... more Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction o...
In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes incl... more In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which ~50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors.
EMB506 is a chloroplast protein essential for embryo development, the function of which is unknow... more EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A twohybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene , Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle.
Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporth... more Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors that are suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1-CO39 that interacts with the rice resistance gene Pi-CO39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR1-CO39 is specifically expressed and secreted at the plant-fungal interface during the biotrophic phase of infection. Live-cell imaging with M. oryzae transformants expressing a translational fusion between AVR1-CO39 and the monomeric red fluorescent protein (mRFP) indicated that AVR1-CO39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1-CO39 isoform without a signal peptide in rice protoplasts triggers a Pi-CO39-specific hypersensitive response, suggesting that recognition of AVR1-CO39 by the Pi-CO39 gene product occurs in the cytoplasm of rice cells. The native AVR1-CO39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1-CO39:mRFP:HDEL translational fusions, and is correctly processed as shown by Western blotting. However, this secreted AVR1-CO39 isoform triggers a Pi-CO39-specific hypersensitive response and accumulates inside rice protoplasts as shown by Western blotting and localization of AVR1-CO39:mRFP translational fusions. This indicates that AVR1-CO39 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR1-CO39 into rice cells occurs independently of fungal factors.
Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect bindin... more Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.
The hypersensitive response (HR) is a programmed cell death that is commonly associated with plan... more The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles. ; fax 33-05-61-28-50-61. Article, publication date, and citation information can be found at www.plantcell.org/cgi/
The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance i... more The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance in an agricultural context has led to the search for genes underlying quantitative resistance in plants. Such genes have been identified in only a few cases, all for fungal or nematode resistance, and encode diverse molecular functions. However, an understanding of the molecular mechanisms of quantitative resistance variation to other enemies and the associated evolutionary forces shaping this variation remain largely unknown. We report the identification, map-based cloning and functional validation of QRX3 (RKS1, Resistance related KinaSe 1), conferring broad-spectrum resistance to Xanthomonas campestris (Xc), a devastating worldwide bacterial vascular pathogen of crucifers. RKS1 encodes an atypical kinase that mediates a quantitative resistance mechanism in plants by restricting bacterial spread from the infection site. Nested Genome-Wide Association mapping revealed a major locus corresponding to an allelic series at RKS1 at the species level. An association between variation in resistance and RKS1 transcription was found using various transgenic lines as well as in natural accessions, suggesting that regulation of RKS1 expression is a major component of quantitative resistance to Xc. The co-existence of long lived RKS1 haplotypes in A. thaliana is shared with a variety of genes involved in pathogen recognition, suggesting common selective pressures. The identification of RKS1 constitutes a starting point for deciphering the mechanisms underlying broad spectrum quantitative disease resistance that is effective against a devastating and vascular crop pathogen. Because putative RKS1 orthologous have been found in other Brassica species, RKS1 provides an exciting opportunity for plant breeders to improve resistance to black rot in crops.
The hxc1 mutant was identified by screening an EMS (ethylmethane sulphonate) mutagenized populati... more The hxc1 mutant was identified by screening an EMS (ethylmethane sulphonate) mutagenized population of Arabidopsis Col-0 plants for an altered hypersensitive response (HR), after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv . campestris ( Xcc ) (strain 147). The hxc1 mutant shows a susceptible phenotype several days after initiation of the interaction with the avirulent strain. This macroscopically observed phenotype was confirmed by measurement of in planta bacterial growth and by microscopical analysis. Interestingly, the hxc1 mutation acts very specifically. Hxc1 displays a pathophenotype identical to that observed in the wild-type with several extensively characterized avirulent and virulent bacteria, except in response to Pseudomonas syringae pv . tomato strain DC3000/ avrRpm1 , for which a partial loss of resistance was observed. Finally, the mutation causes an attenuation of expression of several defence markers regulated through different signalling pathways. Together, these data underline the complexity of this novel defence mutant, and support the hypothesis of a mutation affecting a key component acting during the first steps of the plant defence response leading to resistance to Xcc147 and Pseudomonas syringae pv . tomato containing the avr gene , avrRpm1 .
Plants are continuously exposed to attack by potential phytopathogens. Disease prevention require... more Plants are continuously exposed to attack by potential phytopathogens. Disease prevention requires pathogen recognition and the induction of a multifaceted defense response. We are studying the non-host disease resistance response of parsley to the oomycete, Phytophthora sojae using a cell culture-based system. Receptor-mediated recognition of P. sojae may be achieved through a thirteen amino acid peptide sequence (Pep-13) present within an abundant cell wall transglutaminase. Following recognition of this elicitor molecule, parsley cells mount a defense response, which includes the generation of reactive oxygen species (ROS) and transcriptional activation of genes encoding pathogenesis-related (PR) proteins or enzymes involved in the synthesis of antimicrobial phytoalexins. Treatment of parsley cells with the NADPH oxidase inhibitor, diphenylene iodonium (DPI), blocked both Pep-13-induced phytoalexin production and the accumulation of transcripts encoding enzymes involved in their synthesis. In contrast, DPI treatment had no effect upon Pep-13-induced PR gene expression, suggesting the existence of an oxidative burst-independent mechanism for the transcriptional activation of PR genes. The use of specific antibodies enabled the identification of three parsley mitogen-activated protein kinases (MAPKs) that are activated within the signal transduction pathway(s) triggered following recognition of Pep-13. Other environmental challenges failed to activate these kinases in parsley cells, suggesting that their activation plays a key role in defense signal transduction. Moreover, by making use of a protoplast co-transfection system overexpressing wild-type and loss-of-function MAPK mutants, we show an essential role for post-translational phosphorylation and activation of MAPKs for oxidative burst-independent PR promoter activation.
including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 enc... more including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC Xcc8004 , functions as an avirulence gene whose product seems to be recognized in vascular tissues.
The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the A... more The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the ABA-dependent induction of late embryogenesis abundant (LEA) genes in the ¢nal stages of seed development. We tested whether the VP16 transcriptional activation domain is su⁄cient to provide ABI5 with the ability to activate the AtEm LEA genes in vegetative tissues. We took advantage of a new transgenic seed selection assay based on green £uorescent protein (GFP) £uorescence and found that VP16-ABI5 triggered growth retardation and ABAindependent induction of AtEm1 in seedlings. These results indicate that ABI5 activation potential is a limiting step and might be a target for ABA signaling. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
Cultured parsley (Petroselinum crispum) cells respond to treatment with elicitors derived from di... more Cultured parsley (Petroselinum crispum) cells respond to treatment with elicitors derived from different species of the genus Phytophthora with transcript accumulation of defense-associated genes and the production of furanocoumarin phytoalexins. Pep-25, an oligopeptide fragment of a Phytophthora sojae 42-kDa cell wall protein, and a cell wall elicitor preparation derived from Phytophthora parasitica (Pp-elicitor) stimulate accumulation of the same gene transcripts and formation of the same pattern of furanocoumarins. , respectively. The signature of the Ca 2+ response differed greatly between the two elicitors tested. Extracellular Ca 2+ proved essential for activation of an oxidative burst, MAP kinase activity and phytoalexin production by either elicitor. While Ppelicitor induced a qualitatively similar spectrum of defense responses as did Pep-25, elicitor-specific quantitative differences in response intensity and kinetics suggest activation of a conserved signaling cascade through separate ligand binding sites.
Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and... more Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolat...
Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds... more Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the Magnaporthe oryzae effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from M. oryzae, and ToxB, an effector of the wheat tan spot pathogen Pyrenophora tritici-repentis have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (Magnaporthe Avrs and ToxB like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in M. oryzae and M. grisea where they seem to be particularly important since they account for 5-10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of M. oryzae MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families.
Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and... more Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolates and host-specific lineages, the genome composition in terms of frequencies of genes putatively involved in pathogenicity (effectors, secondary metabolism, cazome) was conserved. However, 529 shared families were found only in nonrice lineages, whereas the rice lineage possessed 86 specific families absent from the nonrice genomes. Our results confirmed that the host specificity of M. oryzae isolates was associated with a divergence between lineages without major gene flow and that, despite the strong conservation of gene families between lineages, adaptation to different hosts, especially to rice, was associated with the presence of a small number of specific gene families. All information was gathered in a public database (http://genome.jouy.inra.fr/gemo).
Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellu... more Plant immunity is often triggered by the specific recognition of pathogen effectors by intracellular nucleotide-binding, leucine-rich repeat receptors (NLR). Plant NLRs contain an N-terminal signaling domain that is mostly represented by either a Toll-interleukin1 receptor (TIR) domain or a coiled coil (CC) domain. In many cases, single NLR proteins are sufficient for both effector recognition and signaling activation. However, many paired NLRs have now been identified where both proteins are required to confer resistance to pathogens. Recent detailed studies on the Arabidopsis thaliana TIR-NLR pair RRS1 and RPS4 and on the rice CC-NLR pair RGA4 and RGA5 have revealed for the first time how such protein pairs function together. In both cases, the paired partners interact physically to form a hetero-complex receptor in which each partner plays distinct roles in effector recognition or signaling activation, highlighting a conserved mode of action of NLR pairs across both monocotyledon...
Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain prote... more Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction o...
In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes incl... more In plants, basic region/leucine zipper motif (bZIP) transcription factors regulate processes including pathogen defence, light and stress signalling, seed maturation and flower development. The Arabidopsis genome sequence contains 75 distinct members of the bZIP family, of which ~50 are not described in the literature. Using common domains, the AtbZIP family can be subdivided into ten groups. Here, we review the available data on bZIP functions in the context of subgroup membership and discuss the interacting proteins. This integration is essential for a complete functional characterization of bZIP transcription factors in plants, and to identify functional redundancies among AtbZIP factors.
EMB506 is a chloroplast protein essential for embryo development, the function of which is unknow... more EMB506 is a chloroplast protein essential for embryo development, the function of which is unknown. A twohybrid interaction screen was performed to provide insight into the role of EMB506. A single interacting partner, AKRP, was identified among a cDNA library from immature siliques. The AKR gene , Plant Cell 4, 1575-1588) encodes a protein containing five ankyrin repeats, very similar to EMB506. Protein truncation series demonstrated that both proteins interact through their ankyrin domains. Using reverse genetics, we showed that loss of akr function resulted in an embryo-defective (emb) phenotype indistinguishable from the emb506 phenotype. Transient expression of the signal peptide of AKRP fused to green fluorescent protein demonstrated the chloroplast localization of AKRP. The ABI3 promoter was used to express AKR in a seed-specific manner in order to analyse the post-embryonic effect of AKR loss of function in akr/akr seedlings. Homozygous fertile and viable akr/akr plants were obtained. These plants exhibited mild to severe defects in chloroplast and leaf cellular organization. We conclude that EMB506 and AKRP are involved in crucial and tightly controlled events in plastid differentiation linked to cell differentiation, morphogenesis and organogenesis during the plant life cycle.
Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporth... more Effector proteins are key elements in plant-fungal interactions. The rice blast fungus Magnaporthe oryzae secretes numerous effectors that are suspected to be translocated inside plant cells. However, their cellular targets and the mechanisms of translocation are still unknown. Here, we have identified the open reading frame (ORF3) corresponding to the M. oryzae avirulence gene AVR1-CO39 that interacts with the rice resistance gene Pi-CO39 and encodes a small secreted protein without homology to other proteins. We demonstrate that AVR1-CO39 is specifically expressed and secreted at the plant-fungal interface during the biotrophic phase of infection. Live-cell imaging with M. oryzae transformants expressing a translational fusion between AVR1-CO39 and the monomeric red fluorescent protein (mRFP) indicated that AVR1-CO39 is translocated into the cytoplasm of infected rice cells. Transient expression of an AVR1-CO39 isoform without a signal peptide in rice protoplasts triggers a Pi-CO39-specific hypersensitive response, suggesting that recognition of AVR1-CO39 by the Pi-CO39 gene product occurs in the cytoplasm of rice cells. The native AVR1-CO39 protein enters the secretory pathway of rice protoplasts as demonstrated by the ER localization of AVR1-CO39:mRFP:HDEL translational fusions, and is correctly processed as shown by Western blotting. However, this secreted AVR1-CO39 isoform triggers a Pi-CO39-specific hypersensitive response and accumulates inside rice protoplasts as shown by Western blotting and localization of AVR1-CO39:mRFP translational fusions. This indicates that AVR1-CO39 is secreted by rice protoplasts and re-enters into the cytoplasm by unknown mechanisms, suggesting that translocation of AVR1-CO39 into rice cells occurs independently of fungal factors.
Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect bindin... more Resistance (R) proteins recognize pathogen avirulence (Avr) proteins by direct or indirect binding and are multidomain proteins generally carrying a nucleotide binding (NB) and a leucine-rich repeat (LRR) domain. Two NB-LRR protein-coding genes from rice (Oryza sativa), RGA4 and RGA5, were found to be required for the recognition of the Magnaporthe oryzae effector AVR1-CO39. RGA4 and RGA5 also mediate recognition of the unrelated M. oryzae effector AVR-Pia, indicating that the corresponding R proteins possess dual recognition specificity. For RGA5, two alternative transcripts, RGA5-A and RGA5-B, were identified. Genetic analysis showed that only RGA5-A confers resistance, while RGA5-B is inactive. Yeast two-hybrid, coimmunoprecipitation, and fluorescence resonance energy transfer-fluorescence lifetime imaging experiments revealed direct binding of AVR-Pia and AVR1-CO39 to RGA5-A, providing evidence for the recognition of multiple Avr proteins by direct binding to a single R protein. Direct binding seems to be required for resistance as an inactive AVR-Pia allele did not bind RGA5-A. A small Avr interaction domain with homology to the Avr recognition domain in the rice R protein Pik-1 was identified in the C terminus of RGA5-A. This reveals a mode of Avr protein recognition through direct binding to a novel, non-LRR interaction domain.
The hypersensitive response (HR) is a programmed cell death that is commonly associated with plan... more The hypersensitive response (HR) is a programmed cell death that is commonly associated with plant disease resistance. A novel lesion mimic mutant, vad1 (for vascular associated death1), that exhibits light conditional appearance of propagative HR-like lesions along the vascular system was identified. Lesion formation is associated with expression of defense genes, production of high levels of salicylic acid (SA), and increased resistance to virulent and avirulent strains of Pseudomonas syringae pv tomato. Analyses of the progeny from crosses between vad1 plants and either nahG transgenic plants, sid1, nonexpressor of PR1 (npr1), enhanced disease susceptibility1 (eds1), or non-race specific disease resistance1 (ndr1) mutants, revealed the vad1 cell death phenotype to be dependent on SA biosynthesis but NPR1 independent; in addition, both EDS1 and NDR1 are necessary for the proper timing and amplification of cell death as well as for increased resistance to Pseudomonas strains. VAD1 encodes a novel putative membrane-associated protein containing a GRAM domain, a lipid or protein binding signaling domain, and is expressed in response to pathogen infection at the vicinity of the hypersensitive lesions. VAD1 might thus represent a new potential function in cell death control associated with cells in the vicinity of vascular bundles. ; fax 33-05-61-28-50-61. Article, publication date, and citation information can be found at www.plantcell.org/cgi/
The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance i... more The failure of gene-for-gene resistance traits to provide durable and broad-spectrum resistance in an agricultural context has led to the search for genes underlying quantitative resistance in plants. Such genes have been identified in only a few cases, all for fungal or nematode resistance, and encode diverse molecular functions. However, an understanding of the molecular mechanisms of quantitative resistance variation to other enemies and the associated evolutionary forces shaping this variation remain largely unknown. We report the identification, map-based cloning and functional validation of QRX3 (RKS1, Resistance related KinaSe 1), conferring broad-spectrum resistance to Xanthomonas campestris (Xc), a devastating worldwide bacterial vascular pathogen of crucifers. RKS1 encodes an atypical kinase that mediates a quantitative resistance mechanism in plants by restricting bacterial spread from the infection site. Nested Genome-Wide Association mapping revealed a major locus corresponding to an allelic series at RKS1 at the species level. An association between variation in resistance and RKS1 transcription was found using various transgenic lines as well as in natural accessions, suggesting that regulation of RKS1 expression is a major component of quantitative resistance to Xc. The co-existence of long lived RKS1 haplotypes in A. thaliana is shared with a variety of genes involved in pathogen recognition, suggesting common selective pressures. The identification of RKS1 constitutes a starting point for deciphering the mechanisms underlying broad spectrum quantitative disease resistance that is effective against a devastating and vascular crop pathogen. Because putative RKS1 orthologous have been found in other Brassica species, RKS1 provides an exciting opportunity for plant breeders to improve resistance to black rot in crops.
The hxc1 mutant was identified by screening an EMS (ethylmethane sulphonate) mutagenized populati... more The hxc1 mutant was identified by screening an EMS (ethylmethane sulphonate) mutagenized population of Arabidopsis Col-0 plants for an altered hypersensitive response (HR), after spray inoculation with an HR-inducing isolate of Xanthomonas campestris pv . campestris ( Xcc ) (strain 147). The hxc1 mutant shows a susceptible phenotype several days after initiation of the interaction with the avirulent strain. This macroscopically observed phenotype was confirmed by measurement of in planta bacterial growth and by microscopical analysis. Interestingly, the hxc1 mutation acts very specifically. Hxc1 displays a pathophenotype identical to that observed in the wild-type with several extensively characterized avirulent and virulent bacteria, except in response to Pseudomonas syringae pv . tomato strain DC3000/ avrRpm1 , for which a partial loss of resistance was observed. Finally, the mutation causes an attenuation of expression of several defence markers regulated through different signalling pathways. Together, these data underline the complexity of this novel defence mutant, and support the hypothesis of a mutation affecting a key component acting during the first steps of the plant defence response leading to resistance to Xcc147 and Pseudomonas syringae pv . tomato containing the avr gene , avrRpm1 .
Plants are continuously exposed to attack by potential phytopathogens. Disease prevention require... more Plants are continuously exposed to attack by potential phytopathogens. Disease prevention requires pathogen recognition and the induction of a multifaceted defense response. We are studying the non-host disease resistance response of parsley to the oomycete, Phytophthora sojae using a cell culture-based system. Receptor-mediated recognition of P. sojae may be achieved through a thirteen amino acid peptide sequence (Pep-13) present within an abundant cell wall transglutaminase. Following recognition of this elicitor molecule, parsley cells mount a defense response, which includes the generation of reactive oxygen species (ROS) and transcriptional activation of genes encoding pathogenesis-related (PR) proteins or enzymes involved in the synthesis of antimicrobial phytoalexins. Treatment of parsley cells with the NADPH oxidase inhibitor, diphenylene iodonium (DPI), blocked both Pep-13-induced phytoalexin production and the accumulation of transcripts encoding enzymes involved in their synthesis. In contrast, DPI treatment had no effect upon Pep-13-induced PR gene expression, suggesting the existence of an oxidative burst-independent mechanism for the transcriptional activation of PR genes. The use of specific antibodies enabled the identification of three parsley mitogen-activated protein kinases (MAPKs) that are activated within the signal transduction pathway(s) triggered following recognition of Pep-13. Other environmental challenges failed to activate these kinases in parsley cells, suggesting that their activation plays a key role in defense signal transduction. Moreover, by making use of a protoplast co-transfection system overexpressing wild-type and loss-of-function MAPK mutants, we show an essential role for post-translational phosphorylation and activation of MAPKs for oxidative burst-independent PR promoter activation.
including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 enc... more including Arabidopsis thaliana. The gene XC1553 from X. campestris pv. campestris strain 8004 encodes a protein containing leucine-rich repeats (LRRs) and appears to be restricted to strains of X. campestris pv. campestris. LRRs are found in a number of type III-secreted effectors in plant and animal pathogens. These prompted us to investigate the role of the XC1553 gene in the interaction between X. campestris pv. campestris and A. thaliana. Translocation assays using the hypersensitive-reaction-inducing domain of X. campestris pv. campestris AvrBs1 as a reporter revealed that XC1553 is a type III effector. Infiltration of Arabidopsis leaf mesophyll with bacterial suspensions showed no differences between the wild-type strain and an XC1553 gene mutant; both strains induced disease symptoms on Kashmir and Col-0 ecotypes. However, a clear difference was observed when bacteria were introduced into the vascular system by piercing the central vein of leaves. In this case, the wild-type strain 8004 caused disease on the Kashmir ecotype, but not on ecotype Col-0; the XC1553 gene mutant became virulent on the Col-0 ecotype and still induced disease on the Kashmir ecotype. Altogether, these data show that the XC1553 gene, which was renamed avrAC Xcc8004 , functions as an avirulence gene whose product seems to be recognized in vascular tissues.
The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the A... more The Arabidopsis abscisic acid (ABA) insensitive (ABI)5 transcription factor participates in the ABA-dependent induction of late embryogenesis abundant (LEA) genes in the ¢nal stages of seed development. We tested whether the VP16 transcriptional activation domain is su⁄cient to provide ABI5 with the ability to activate the AtEm LEA genes in vegetative tissues. We took advantage of a new transgenic seed selection assay based on green £uorescent protein (GFP) £uorescence and found that VP16-ABI5 triggered growth retardation and ABAindependent induction of AtEm1 in seedlings. These results indicate that ABI5 activation potential is a limiting step and might be a target for ABA signaling. ß 2004 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.
Cultured parsley (Petroselinum crispum) cells respond to treatment with elicitors derived from di... more Cultured parsley (Petroselinum crispum) cells respond to treatment with elicitors derived from different species of the genus Phytophthora with transcript accumulation of defense-associated genes and the production of furanocoumarin phytoalexins. Pep-25, an oligopeptide fragment of a Phytophthora sojae 42-kDa cell wall protein, and a cell wall elicitor preparation derived from Phytophthora parasitica (Pp-elicitor) stimulate accumulation of the same gene transcripts and formation of the same pattern of furanocoumarins. , respectively. The signature of the Ca 2+ response differed greatly between the two elicitors tested. Extracellular Ca 2+ proved essential for activation of an oxidative burst, MAP kinase activity and phytoalexin production by either elicitor. While Ppelicitor induced a qualitatively similar spectrum of defense responses as did Pep-25, elicitor-specific quantitative differences in response intensity and kinetics suggest activation of a conserved signaling cascade through separate ligand binding sites.
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Papers by Thomas Kroj