Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

Identification of Pseudomonas strains for the biological control of soybean red crown root rot.

Soybean red crown root rot (RCR), caused by the soil-borne fungal pathogen, Calonectria ilicicola, is the most destructive disease affecting soybean production in Japan. To date, no resistant cultivars or effective fungicides have been developed to control this disease. In this study, we evaluated 13 bacterial strains to determine their efficacy in controlling C. ilicicola. We first investigated whether the volatile organic compounds (VOCs) emitted by the bacterial strains exhibited any antifungal activity against C. ilicicola using the double-plate chamber method. The results showed that VOCs from three Pseudomonas bacterial strains, OFT2 (Pseudomonas sp.), OFT5 (Pseudomonas sp.), and Cab57 (Pseudomonas protegens), exhibited strong inhibitory activity against C. ilicicola mycelial growth. Some antifungal activity was also observed in the culture supernatants of these Pseudomonas strains. Greenhouse soil inoculation tests showed that application of OFT2, OFT5, and Cab57 cultures around soybean seeds after seed sowing significantly reduced the severity of RCR, as shown by up to 40% reduction in C. ilicicola fungal growth in the roots and 180-200% increase in shoot and root fresh weights compared to the water control. Our results suggest that OFT2, Cab57, and OFT5 produce potent antifungal compounds against C. ilicicola, thereby showing considerable potential for the biological control of C. ilicicola during soybean production.

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution.

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

A jacalin-like lectin domain-containing protein of Sclerospora graminicola acts as an apoplastic virulence effector in plant-oomycete interactions.

The plant extracellular space, including the apoplast and plasma membrane, is the initial site of plant-pathogen interactions. Pathogens deliver numerous secreted proteins, called effectors, into this region to suppress plant immunity and establish infection. Downy mildew caused by the oomycete pathogen Sclerospora graminicola (Sg) is an economically important disease of Poaceae crops including foxtail millet (Setaria italica). We previously reported the genome sequence of Sg and showed that the jacalin-related lectin (JRL) gene family has significantly expanded in this lineage. However, the biological functions of JRL proteins remained unknown. Here, we show that JRL from Sg (SgJRL) functions as an apoplastic virulence effector. We identified eight SgJRLs by protein mass spectrometry analysis of extracellular fluid from Sg-inoculated foxtail millet leaves. SgJRLs consist of a jacalin-like lectin domain and an N-terminal putative secretion signal; SgJRL expression is induced by Sg infection. Heterologous expression of three SgJRLs with N-terminal secretion signal peptides in Nicotiana benthamiana enhanced the virulence of the pathogen Phytophthora palmivora inoculated onto the same leaves. Of the three SgJRLs, SG06536 fused with green fluorescent protein (GFP) localized to the apoplastic space in N. benthamiana leaves. INF1-mediated induction of defence-related genes was suppressed by co-expression of SG06536-GFP. These findings suggest that JRLs are novel apoplastic effectors that contribute to pathogenicity by suppressing plant defence responses.

Recombinant inbred lines and next-generation sequencing enable rapid identification of candidate genes involved in morphological and agronomic traits in foxtail millet.

We constructed recombinant inbred lines (RILs) between a Japanese and a Taiwanese landrace of foxtail millet and employed next-generation sequencing, such as flexible ddRAD-seq and Nanopore sequencing to identify the candidate genes involved in the crop evolution of foxtail millet. We successfully constructed a linkage map using flexible ddRAD-seq with parents and RILs and detected major QTLs for each of three traits: leaf sheath colors, spikelet-tipped bristles (stb), and days to heading (DTH). (1) For leaf sheath colors, we identified the C gene on chromosome IV. (2) We identified a homeobox (HOX14) gene for stb on chromosome II, which shows homology with HvVrs1 in barley. (3) Finally, we identified a QTL with a large effect on DTH on chromosome II. A parent of the RILs from Taiwan and Yugu1 had a Harbinger-like TE in intron 3 of this gene. We also investigated the geographical distribution of the TE insertion type of this gene and found that the insertion type is distributed in the northern part of East Asia and intensively in South and Southeast Asia, suggesting that loss/reduction of function of this gene plays an important role in spreading into the northern part of East Asia and subtropical and tropical zones.

Soybean Hypocotyls Prevent Calonectria ilicicola Invasion by Multi-Layered Defenses.

In plants, many pathogens infect a specific set of host organs to cause disease, yet the underlying mechanisms remain unclear. Here, we show that inoculation of soybean plants with Calonectria ilicicola, the soil-borne causal agent of soybean red crown rot, caused typical disease symptoms of root rot and leaf chlorosis and necrosis. However, the pathogen DNA was only detected in the roots and stem (hypocotyl) base but not other aerial parts of the plants. As we observed vigorous fungal growth in all culture media made of extracts from roots, stems, and leaves, differences in key components including available nutrients did not determine organ-specific infection and reproduction by C. ilicicola. Furthermore, inoculation of stems both with and without a surface wound showed that the stems resisted C. ilicicola infection via both the pre- and post-invasion defense layers. Transcriptomic comparison of roots and stems using RNA-seq analysis further revealed that upon C. ilicicola inoculation, a greater expression of genes involved in stress response was induced in the plant stems, including receptor-like kinase, AP2/ERF, MYB, and WRKY. In addition, pathways related to amino acid metabolism were also more upregulated in the stems in response to C. ilicicola infection. These results suggest that soybean stems provide C. ilicicola resistance, at least in part, by activating an organ-specific defense response.

RNA-Seq of in planta-expressed Magnaporthe oryzae genes identifies MoSVP as a highly expressed gene required for pathogenicity at the initial stage of infection.

The ascomycete fungus Magnaporthe oryzae is a hemibiotrophic pathogen that causes rice blast disease. Magnaporthe oryzae infects rice leaves, stems and panicles, and induces severe reductions in yield. Effector proteins secreted by M. oryzae in planta are thought to be involved its virulence activity. Here, using RNA-sequencing (RNA-Seq), we generated transcriptome data for M. oryzae isolate Ina168 during the initial stages of infection. We prepared samples from conidia (the inoculum) and from peeled epidermal cotyledon tissue of susceptible barley Hordeum vulgare 'Nigrate' at 12, 24, 36 and 48 hours post-inoculation (hpi). We also generated a draft genome sequence of M. oryzae isolate Ina168 and used it as a reference for mapping the RNA-Seq reads. Gene expression profiling across all stages of M. oryzae infection revealed 1728 putative secreted effector protein genes. We selected seven such genes that were strongly up-regulated at 12 hpi and down-regulated at 24 or 36 hpi and performed gene knockout analysis to determine their roles in pathogenicity. Knockout of MoSVP, encoding a small putative secreted protein with a hydrophobic surface binding protein A domain, resulted in a reduction in pathogenicity, suggesting that MoSVP is a novel virulence effector of M. oryzae.

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

BACKGROUND: Downy 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. RESULTS: Sequence 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. CONCLUSIONS: We 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.

Chlorella ingestion suppresses resistin gene expression in peripheral blood cells of borderline diabetics.

BACKGROUND & AIMS: Type 2 diabetes can lead to arteriosclerosis, renal damage, retinopathy, and several other serious complications. To prevent or delay the progression of this disease, considerable attention has been paid to improve exercise and dietary habits. Chlorella ingestion can reportedly reduce high blood glucose and cholesterol levels in mice and humans, although no studies have critically evaluated the effects of Chlorella on human borderline diabetics. Thus, we conducted a randomized, double-blind placebo-controlled test with volunteer borderline diabetics. METHODS: We recruited 57 subjects and randomly divided into a Chlorella ingestion group (n = 28) and a Placebo ingestion group (n = 29). Blood samples were collected every 4 weeks for laboratory tests. Gene expression analyses using peripheral blood cell RNA were performed before and 12 weeks after the trial. RESULTS: A total of 252 genes showed changed expression levels between these two groups. Six of these were type 2 diabetes-associated genes, including resistin, an insulin resistance inducer that exhibited markedly reduced expression with Chlorella ingestion (P = 0.01). Resistin mRNA expression significantly correlated with changes in HbA1c and TNF-α and IL-6 levels, all of which are strongly associated with glucose metabolism and/or inflammation. CONCLUSIONS: Chlorella ingestion may be useful in preventing or ameliorating the course of type 2 diabetes development. In addition, gene expression analysis may be a means to investigate the effects of foods and supplements in humans.

Jasmonic acid negatively regulates resistance to Tobacco mosaic virus in tobacco.

Nicotiana tabacum (tobacco) cultivars possessing the N resistance gene to Tobacco mosaic virus (TMV) induce a hypersensitive response, which is accompanied by the production of phytohormones such as salicylic acid (SA) and jasmonic acid (JA), to enclose the invaded virus at the initial site of infection, which inhibits viral multiplication and spread. SA functions as a positive regulator of TMV resistance. However, the role of JA in TMV resistance has not been fully elucidated. Exogenously applied methyl jasmonate, a methyl ester of JA, reduced local resistance to TMV and permitted systemic viral movement. Furthermore, in contrast to a previous finding, we demonstrated that silencing of CORONATINE-INSENSITIVE 1 (COI1), a JA receptor, reduced viral accumulation in a tobacco cultivar possessing the N gene, as did that of allene oxide synthase, a JA biosynthetic enzyme. The reduction in viral accumulation in COI1-silenced tobacco plants was correlated with an increase in SA, and lowering SA levels by introducing an SA hydroxylase gene attenuated this reduction. Viral susceptibility did not change in a COI1-silenced tobacco cultivar lacking the N gene. These results suggest that JA signaling is not directly responsible for susceptibility to TMV, but is indirectly responsible for viral resistance through the partial inhibition of SA-mediated resistance conferred by the N gene, and that a balance between endogenous JA and SA levels is important for determining the degree of resistance.

The variable domain of a plant calcium-dependent protein kinase (CDPK) confers subcellular localization and substrate recognition for NADPH oxidase.

Calcium-dependent protein kinases (CDPKs) are Ca(2+) sensors that regulate diverse biological processes in plants and apicomplexans. However, how CDPKs discriminate specific substrates in vivo is still largely unknown. Previously, we found that a potato StCDPK5 is dominantly localized to the plasma membrane and activates the plasma membrane NADPH oxidase (RBOH; for respiratory burst oxidase homolog) StRBOHB by direct phosphorylation of the N-terminal region. Here, we report the contribution of the StCDPK5 N-terminal variable (V) domain to activation of StRBOHB in vivo using heterologous expression system in Nicotiana benthamiana. Mutations of N-terminal myristoylation and palmitoylation sites in the V domain eliminated the predominantly plasma membrane localization and the capacity of StCDPK5 to activate StRBOHB in vivo. A tomato SlCDPK2, which also contains myristoylation and palmitoylation sites in its N terminus, phosphorylated StRBOHB in vitro but not in vivo. Functional domains responsible for activation and phosphorylation of StRBOHB were identified by swapping regions for each domain between StCDPK5 and SlCDPK2. The substitution of the V domain of StCDPK5 with that of SlCDPK2 abolished the activation and phosphorylation abilities of StRBOHB in vivo and relocalized the chimeric CDPK to the trans-Golgi network, as observed for SlCDPK2. Conversely, SlCDPK2 substituted with the V domain of StCDPK5 localized to the plasma membrane and activated StRBOHB. These results suggest that the V domains confer substrate specificity in vivo by dictating proper subcellular localization of CDPKs.

StCDPK5 confers resistance to late blight pathogen but increases susceptibility to early blight pathogen in potato via reactive oxygen species burst.

• Potato (Solanum tuberosum) calcium-dependent protein kinase (StCDPK5) has been shown to phosphorylate the N-terminal region of plasma membrane RBOH (respiratory burst oxidase homolog) proteins, and participate in StRBOHB-mediated reactive oxygen species (ROS) burst. The constitutively active form, StCDPK5VK, provides a useful tool for gain-of-function analysis of RBOH in defense responses. • StCDPK5- and StCDPK5VK-green fluorescent protein fusion proteins were predominantly targeted to the plasma membrane, and conditional expression of StCDPK5VK activated StRBOHA-D. The interaction was confirmed by bimolecular fluorescence complementation assay. We generated transgenic potato plants containing StCDPK5VK under the control of a pathogen-inducible promoter to investigate the role of ROS burst on defense responses to blight pathogens. • Virulent isolates of the late blight pathogen Phytophthora infestans and the early blight pathogen Alternaria solani induced hypersensitive response-like cell death accompanied by ROS production at the infection sites of transgenic plants. Transgenic plants showed resistance to the near-obligate hemibiotrophic pathogen P. infestans and, by contrast, increased susceptibility to the necrotrophic pathogen A. solani. • These results indicate that RBOH-dependent ROS contribute to basal defense against near-obligate pathogens, but have a negative role in resistance or have a positive role in expansion of disease lesions caused by necrotrophic pathogens.

Identification of natural diterpenes that inhibit bacterial wilt disease in tobacco, tomato and Arabidopsis.

The soil-borne bacterial pathogen Ralstonia solanacearum invades a broad range of plants through their roots, resulting in wilting of the plant, but no effective protection against this disease has been developed. Two bacterial wilt disease-inhibiting compounds were biochemically isolated from tobacco and identified as sclareol and cis-abienol, labdane-type diterpenes. When exogenously applied to their roots, sclareol and cis-abienol inhibited wilt disease in tobacco, tomato and Arabidopsis plants without exhibiting any antibacterial activity. Microarray analysis identified many sclareol-responsive genes in Arabidopsis roots, including genes encoding or with a role in ATP-binding cassette (ABC) transporters, and biosynthesis and signaling of defense-related molecules and mitogen-activated protein kinase (MAPK) cascade components. Inhibition of wilt disease by sclareol was attenuated in Arabidopsis mutants defective in the ABC transporter AtPDR12, the MAPK MPK3, and ethylene and abscisic acid signaling pathways, and also in transgenic tobacco plants with reduced expression of NtPDR1, a tobacco homolog of AtPDR12. These results suggest that multiple host factors are involved in the inhibition of bacterial wilt disease by sclareol-related compounds.

A rice calcium-dependent protein kinase OsCPK12 oppositely modulates salt-stress tolerance and blast disease resistance.

Calcium-dependent protein kinases (CDPKs) regulate the downstream components in calcium signaling pathways. We investigated the effects of overexpression and disruption of an Oryza sativa (rice) CDPK (OsCPK12) on the plant's response to abiotic and biotic stresses. OsCPK12-overexpressing (OsCPK12-OX) plants exhibited increased tolerance to salt stress. The accumulation of hydrogen peroxide (H(2) O(2) ) in the leaves was less in OsCPK12-OX plants than in wild-type (WT) plants. Genes encoding reactive oxygen species (ROS) scavenging enzymes (OsAPx2 and OsAPx8) were more highly expressed in OsCPK12-OX plants than in WT plants, whereas the expression of the NADPH oxidase gene, OsrbohI, was decreased in OsCPK12-OX plants compared with WT plants. Conversely, a retrotransposon (Tos17) insertion mutant, oscpk12, and plants transformed with an OsCPK12 RNA interference (RNAi) construct were more sensitive to high salinity than were WT plants. The level of H(2) O(2) accumulation was greater in oscpk12 and OsCPK12 RNAi plants than in the WT. These results suggest that OsCPK12 promotes tolerance to salt stress by reducing the accumulation of ROS. We also observed that OsCPK12-OX seedlings had increased sensitivity to abscisic acid (ABA) and increased susceptibility to blast fungus, probably resulting from the repression of ROS production and/or the involvement of OsCPK12 in the ABA signaling pathway. Collectively, our results suggest that OsCPK12 functions in multiple signaling pathways, positively regulating salt tolerance and negatively modulating blast resistance.

Identification of an amino acid residue required for differential recognition of a viral movement protein by the Tomato mosaic virus resistance gene Tm-2(2).

The Tm-2 gene of tomato and its allelic gene, Tm-2(2), confer resistance to Tomato mosaic virus (ToMV) and encode a member of the coiled-coil/nucleotide binding-ARC/leucine-rich repeat (LRR) protein class of plant resistance (R) genes. Despite exhibiting only four amino acid differences between the products of Tm-2 and Tm-2(2), Tm-2(2) confers resistance to ToMV mutant B7, whereas Tm-2 is broken by ToMV-B7. An Agrobacterium-mediated transient expression system was used to study the mechanism of differential recognition of the movement proteins (MPs), an avirulence factor for ToMV resistance, of ToMV-B7 by Tm-2 and Tm-2(2). Although resistance induced by Tm-2 and Tm-2(2) is not usually accompanied by hypersensitive response (HR), Tm-2 and Tm-2(2) induced HR-like cell death by co-expression with MP of a wild-type ToMV, a strain that causes resistance for these R genes, and Tm-2(2) but not Tm-2 induced cell death with B7-MP in this system. Site-directed amino acid mutagenesis revealed that Tyr-767 in the LRR of Tm-2(2) is required for the specific recognition of the B7-MP. These results suggest that the Tyr residue in LRR contributes to the recognition of B7-MP, and that Tm-2 and Tm-2(2) are involved in HR cell death.

Regulatory mechanisms of nitric oxide and reactive oxygen species generation and their role in plant immunity.

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in diverse physiological processes, such as programmed cell death, development, cell elongation and hormonal signaling, in plants. Much attention has been paid to the regulation of plant innate immunity by these signal molecules. Recent studies provide evidence that an NADPH oxidase, respiratory burst oxidase homolog, is responsible for pathogen-responsive ROS burst. However, we still do not know about NO-producing enzymes, except for nitrate reductase, although many studies suggest the existence of NO synthase-like activity responsible for NO burst in plants. Here, we introduce regulatory mechanisms of NO and ROS bursts by mitogen-activated protein kinase cascades, calcium-dependent protein kinase or riboflavin and its derivatives, flavin mononucleotide and flavin adenine dinucleotide, and we discuss the roles of the bursts in defense responses against plant pathogens.

Influence of Chlorella powder intake during swimming stress in mice.

We used the forced swimming test to investigate the influence of Chlorella powder intake during muscle stress training in mice. After day 14, swimming time was about 2-fold longer for Chlorella intake mice than for control swimming mice. Microarray analysis revealed that the global gene expression profile of muscle from the Chlorella intake mice was similar to that of muscle from the intact (non-swimming) mice, and the profile of these two groups differed from that of the control (swimming) mice. Gene ontology and pathway analyses of gene expression data showed that oxidoreductase activity and the leukotriene synthesis pathway were repressed in the Chlorella intake mice following the swimming test. In addition, measurements of free fatty acids, glucose, triglycerides, and lactic acid in the blood of Chlorella intake mice were higher than that of control mice. These findings suggest that metabolism in tissues is altered by Chlorella intake.

Silencing of WIPK and SIPK mitogen-activated protein kinases reduces tobacco mosaic virus accumulation but permits systemic viral movement in tobacco possessing the N resistance gene.

Infection of tobacco cultivars possessing the N resistance gene with Tobacco mosaic virus (TMV) results in confinement of the virus by necrotic lesions at the infection site. Although the mitogen-activated protein kinases WIPK and SIPK have been implicated in TMV resistance, evidence linking them directly to disease resistance is, as yet, insufficient. Viral multiplication was reduced slightly in WIPK- or SIPK-silenced plants but substantially in WIPK/SIPK-silenced plants, and was correlated with an increase in salicylic acid (SA) and a decrease in jasmonic acid (JA). Silencing of WIPK and SIPK in a tobacco cultivar lacking the N gene did not inhibit viral accumulation. The reduction in viral accumulation was attenuated by expressing a gene for an SA-degrading enzyme or by exogenously applying JA. Inoculation of lower leaves resulted in the systemic spread of TMV and formation of necrotic lesions in uninoculated upper leaves. These results suggested that WIPK and SIPK function to negatively regulate local resistance to TMV accumulation, partially through modulating accumulation of SA and JA in an N-dependent manner, but positively regulate systemic resistance.

Effect of exercise on gene expression profile in unfractionated peripheral blood leukocytes.

A 4-h bout of exercise induces immunomodulatory effects. Peripheral blood was withdrawn before, and at 4, 8 and 24h after the start of exercise. RNA from the unfractionated white blood cells was analyzed using Agilent human 44K microarray. The expression profiles were sorted into seven clusters based on their unique time-dependent kinetics. In a separate experiment, cell-specific markers were collected and compared among the members in each cluster. Two clusters were assigned as representing neutrophils, one as NK cells, and another mostly as T cells. Three clusters seemed to be mixtures of several cell types. Extension of this approach to other systems is discussed.

Molecular mechanisms of generation for nitric oxide and reactive oxygen species, and role of the radical burst in plant immunity.

Rapid production of nitric oxide (NO) and reactive oxygen species (ROS) has been implicated in the regulation of innate immunity in plants. A potato calcium-dependent protein kinase (StCDPK5) activates an NADPH oxidase StRBOHA to D by direct phosphorylation of N-terminal regions, and heterologous expression of StCDPK5 and StRBOHs in Nicotiana benthamiana results in oxidative burst. The transgenic potato plants that carry a constitutively active StCDPK5 driven by a pathogen-inducible promoter of the potato showed high resistance to late blight pathogen Phytophthora infestans accompanied by HR-like cell death and H(2)O(2) accumulation in the attacked cells. In contrast, these plants showed high susceptibility to early blight necrotrophic pathogen Alternaria solani, suggesting that oxidative burst confers high resistance to biotrophic pathogen, but high susceptibility to necrotrophic pathogen. NO and ROS synergistically function in defense responses. Two MAPK cascades, MEK2-SIPK and cytokinesis-related MEK1-NTF6, are involved in the induction of NbRBOHB gene in N. benthamiana. On the other hand, NO burst is regulated by the MEK2-SIPK cascade. Conditional activation of SIPK in potato plants induces oxidative and NO bursts, and confers resistance to both biotrophic and necrotrophic pathogens, indicating the plants may have obtained during evolution the signaling pathway which regulates both NO and ROS production to adapt to wide-spectrum pathogens.