H. Saitoh

Cytosolic HSP90 and HSP70 are essential components of INF1‐mediated hypersensitive response and non‐host resistance to Pseudomonas cichorii in Nicotiana benthamiana

SUMMARY Mitogen-activated protein kinases (MAPKs) play pivotal roles in the signal transduction pathway of plant defence responses against pathogens. A search for MAPK-interacting proteins revealed an interaction between a Nicotiana benthamiana MAPK, SIPK (NbSIPK) and cytosolic Hsp90 (NbHsp90c-1) in yeast two-hybrid assay. To study the function of Hsp90 in disease resistance, we silenced NbHsp90c-1 in N. benthamiana by virus-induced gene silencing (VIGS) with Potato virus X (PVX). NbHsp90c-1 silenced plants exhibited: (1) a stunted phenotype, (2) no hypersensitive response (HR) development after infiltration with the Phytophthora infestans protein INF1 and a non-host pathogen Pseudomonas cichorii that normally triggers HR in N. benthamiana, (3) compromised non-host resistance to P. cichorii, and (4) consistently reduced transcription levels of PR (pathogenesis related) protein genes. Similar phenotypes were observed also for plants in which a cytosolic Hsp70 (NbHsp70c-1), a gene for another class of molecular chaperon, was silenced. Hsp90 was isolated as a MAPK-interacting protein in yeast two-hybrid assay, therefore we tested the effect of NbHsp90c-1 silencing as well as NbHsp70c-1 silencing on the HR development caused by infiltration of a hyperactive potato MAPKK (StMEK1(DD)). No difference in the timing or extent of HR was found among NbHsp90c-1 silenced, NbHsp70c-1 silenced and control plants. This result indicates that observed impairment of INF1- and P. cichorii-mediated HR development in NbHsp90c-1 silenced and NbHsp70c-1 silenced plants was not caused by the abrogation in MAPK function downstream of active MAPKK that leads to HR. These findings suggest essential roles of Hsp90 and Hsp70 in plant defence signal transduction pathway upstream or independent of the MAPK cascade.

Virus-induced silencing of WIPK and SIPK genes reduces resistance to a bacterial pathogen, but has no effect on the INF1-induced hypersensitive response (HR) in Nicotiana benthamiana

Activation of two mitogen-activated protein kinases (MAPKs), wound-induced protein kinase (WIPK) and salicylic acid-induced protein kinase (SIPK), is one of the earliest responses that occur in tobacco plants that have been wounded, treated with pathogen-derived elicitors or challenged with avirulent pathogens. We isolated cDNAs for these MAPKs ( NbWIPK and NbSIPK) from Nicotiana benthamiana. The function of NbWIPK and NbSIPK in mediating the hypersensitive response (HR) triggered by infiltration with INF1 protein (the major elicitin secreted by Phytophthora infestans), and the defense response to an incompatible bacterial pathogen ( Pseudomonas cichorii), was investigated by employing virus-induced gene silencing (VIGS) to inhibit expression of the WIPK and SIPK genes in N. benthamiana. Silencing of WIPK or SIPK, or both genes simultaneously, resulted in reduced resistance to P. cichorii, but no change was observed in the timing or extent of HR development after treatment with INF1.

Structural basis of pathogen recognition by an integrated HMA domain in a plant NLR immune receptor

Plants have evolved intracellular immune receptors to detect pathogen proteins known as effectors. How these immune receptors detect effectors remains poorly understood. Here we describe the structural basis for direct recognition of AVR-Pik, an effector from the rice blast pathogen, by the rice intracellular NLR immune receptor Pik. AVR-PikD binds a dimer of the Pikp-1 HMA integrated domain with nanomolar affinity. The crystal structure of the Pikp-HMA/AVR-PikD complex enabled design of mutations to alter protein interaction in yeast and in vitro, and perturb effector-mediated response both in a rice cultivar containing Pikp and upon expression of AVR-PikD and Pikp in the model plant Nicotiana benthamiana. These data reveal the molecular details of a recognition event, mediated by a novel integrated domain in an NLR, which initiates a plant immune response and resistance to rice blast disease. Such studies underpin novel opportunities for engineering disease resistance to plant pathogens in staple food crops. DOI: http://dx.doi.org/10.7554/eLife.08709.001

Serial Analysis of Gene Expression (SAGE) of Magnaporthe grisea: genes involved in appressorium formation

Treatment with cyclic AMP (cAMP) induces appressorium formation in the phytopathogenic fungus Magnaporthe grisea, the causative agent of rice blast disease. In a search for the M. grisea genes responsible for appressorium formation and host invasion, SAGE (Serial Analysis of Gene Expression) was carried out using mRNA isolated from fungal conidia germinating in the presence and absence of cAMP. From cAMP-treated conidia 5087 tags including 2889 unique tags were isolated, whereas untreated conidia yielded 2342 unique tags out of total of 3938. cAMP treatment resulted in up- and down-regulation of genes corresponding to 57 and 53 unique tags, respectively. Upon consultation of EST/cDNA databases, 22 tags with higher representation in cAMP-treated conidia were annotated with putative gene names. Furthermore, 28 tags corresponding to cAMP-induced genes could be annotated with the help of the recently published genome sequence of M. grisea. cAMP-induced genes identified by SAGE included many genes that have not been described so far, as well as a number of genes known to be involved in pathogenicity, e.g. MPG1, MAS1 and MAC1. RT-PCR of 13 randomly selected genes confirmed the SAGE results, verifying the fidelity of the SAGE data.

Polymorphic residues in rice NLRs expand binding and response to effectors of the blast pathogen

Accelerated adaptive evolution is a hallmark of plant–pathogen interactions. Plant intracellular immune receptors (NLRs) often occur as allelic series with differential pathogen specificities. The determinants of this specificity remain largely unknown. Here, we unravelled the biophysical and structural basis of expanded specificity in the allelic rice NLR Pik, which responds to the effector AVR-Pik from the rice blast pathogen Magnaporthe oryzae. Rice plants expressing the Pikm allele resist infection by blast strains expressing any of three AVR-Pik effector variants, whereas those expressing Pikp only respond to one. Unlike Pikp, the integrated heavy metal-associated (HMA) domain of Pikm binds with high affinity to each of the three recognized effector variants, and variation at binding interfaces between effectors and Pikp-HMA or Pikm-HMA domains encodes specificity. By understanding how co-evolution has shaped the response profile of an allelic NLR, we highlight how natural selection drove the emergence of new receptor specificities. This work has implications for the engineering of NLRs with improved utility in agriculture.NLR immune receptors recognize pathogen effectors and activate a response that leads to resistance. The specific interactions between five rice receptor variants and their cognate effectors are studied by solving the structures of the complexes.

Genome analysis of the foxtail millet pathogen Sclerospora graminicola reveals the complex effector repertoire of graminicolous downy mildews

BackgroundDowny mildew, caused by the oomycete pathogen Sclerospora graminicola, is an economically important disease of Gramineae crops including foxtail millet (Setaria italica). Plants infected with S. graminicola are generally stunted and often undergo a transformation of flower organs into leaves (phyllody or witches’ broom), resulting in serious yield loss. To establish the molecular basis of downy mildew disease in foxtail millet, we carried out whole-genome sequencing and an RNA-seq analysis of S. graminicola.ResultsSequence reads were generated from S. graminicola using an Illumina sequencing platform and assembled de novo into a draft genome sequence comprising approximately 360 Mbp. Of this sequence, 73% comprised repetitive elements, and a total of 16,736 genes were predicted from the RNA-seq data. The predicted genes included those encoding effector-like proteins with high sequence similarity to those previously identified in other oomycete pathogens. Genes encoding jacalin-like lectin-domain-containing secreted proteins were enriched in S. graminicola compared to other oomycetes. Of a total of 1220 genes encoding putative secreted proteins, 91 significantly changed their expression levels during the infection of plant tissues compared to the sporangia and zoospore stages of the S. graminicola lifecycle.ConclusionsWe established the draft genome sequence of a downy mildew pathogen that infects Gramineae plants. Based on this sequence and our transcriptome analysis, we generated a catalog of in planta-induced candidate effector genes, providing a solid foundation from which to identify the effectors causing phyllody.