Rice apoplastic CBM1-interacting protein counters blast pathogen invasion by binding conserved carbohydrate binding module 1 motif of fungal proteins.

When infecting plants, fungal pathogens secrete cell wall-degrading enzymes (CWDEs) that break down cellulose and hemicellulose, the primary components of plant cell walls. Some fungal CWDEs contain a unique domain, named the carbohydrate binding module (CBM), that facilitates their access to polysaccharides. However, little is known about how plants counteract pathogen degradation of their cell walls. Here, we show that the rice cysteine-rich repeat secretion protein OsRMC binds to and inhibits xylanase MoCel10A of the blast fungus pathogen Magnaporthe oryzae, interfering with its access to the rice cell wall and degradation of rice xylan. We found binding of OsRMC to various CBM1-containing enzymes, suggesting that it has a general role in inhibiting the action of CBM1. OsRMC is localized to the apoplast, and its expression is strongly induced in leaves infected with M. oryzae. Remarkably, knockdown and overexpression of OsRMC reduced and enhanced rice defense against M. oryzae, respectively, demonstrating that inhibition of CBM1-containing fungal enzymes by OsRMC is crucial for rice defense. We also identified additional CBM-interacting proteins (CBMIPs) from Arabidopsis thaliana and Setaria italica, indicating that a wide range of plants counteract pathogens through this mechanism.

Conserved fungal effector suppresses PAMP-triggered immunity by targeting plant immune kinases.

Plant pathogens have optimized their own effector sets to adapt to their hosts. However, certain effectors, regarded as core effectors, are conserved among various pathogens, and may therefore play an important and common role in pathogen virulence. We report here that the widely distributed fungal effector NIS1 targets host immune components that transmit signaling from pattern recognition receptors (PRRs) in plants. NIS1 from two Colletotrichum spp. suppressed the hypersensitive response and oxidative burst, both of which are induced by pathogen-derived molecules, in Nicotiana benthamianaMagnaporthe oryzae NIS1 also suppressed the two defense responses, although this pathogen likely acquired the NIS1 gene via horizontal transfer from Basidiomycota. Interestingly, the root endophyte Colletotrichum tofieldiae also possesses a NIS1 homolog that can suppress the oxidative burst in N. benthamiana We show that NIS1 of multiple pathogens commonly interacts with the PRR-associated kinases BAK1 and BIK1, thereby inhibiting their kinase activities and the BIK1-NADPH oxidase interaction. Furthermore, mutations in the NIS1-targeting proteins, i.e., BAK1 and BIK1, in Arabidopsis thaliana also resulted in reduced immunity to Colletotrichum fungi. Finally, M. oryzae lacking NIS1 displayed significantly reduced virulence on rice and barley, its hosts. Our study therefore reveals that a broad range of filamentous fungi maintain and utilize the core effector NIS1 to establish infection in their host plants and perhaps also beneficial interactions, by targeting conserved and central PRR-associated kinases that are also known to be targeted by bacterial effectors.

The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response.

Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2 Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G-A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants.

Deployment of the Burkholderia glumae type III secretion system as an efficient tool for translocating pathogen effectors to monocot cells.

Genome sequences of plant fungal pathogens have enabled the identification of effectors that cooperatively modulate the cellular environment for successful fungal growth and suppress host defense. Identification and characterization of novel effector proteins are crucial for understanding pathogen virulence and host-plant defense mechanisms. Previous reports indicate that the Pseudomonas syringae pv. tomato DC3000 type III secretion system (T3SS) can be used to study how non-bacterial effectors manipulate dicot plant cell function using the effector detector vector (pEDV) system. Here we report a pEDV-based effector delivery system in which the T3SS of Burkholderia glumae, an emerging rice pathogen, is used to translocate the AVR-Pik and AVR-Pii effectors of the fungal pathogen Magnaporthe oryzae to rice cytoplasm. The translocated AVR-Pik and AVR-Pii showed avirulence activity when tested in rice cultivars containing the cognate R genes. AVR-Pik reduced and delayed the hypersensitive response triggered by B. glumae in the non-host plant Nicotiana benthamiana, indicative of an immunosuppressive virulence activity. AVR proteins fused with fluorescent protein and nuclear localization signal were delivered by B. glumae T3SS and observed in the nuclei of infected cells in rice, wheat, barley and N. benthamiana. Our bacterial T3SS-enabled eukaryotic effector delivery and subcellular localization assays provide a useful method for identifying and studying effector functions in monocot plants.

Functional differentiation in the leucine-rich repeat domains of closely related plant virus-resistance proteins that recognize common avr proteins.

The N' gene of Nicotiana sylvestris and L genes of Capsicum plants confer the resistance response accompanying the hypersensitive response (HR) elicited by tobamovirus coat proteins (CP) but with different viral specificities. Here, we report the identification of the N' gene. We amplified and cloned an N' candidate using polymerase chain reaction primers designed from L gene sequences. The N' candidate gene was a single 4143 base pairs fragment encoding a coiled-coil nucleotide-binding leucine-rich repeat (LRR)-type resistance protein of 1,380 amino acids. The candidate gene induced the HR in response to the coexpression of tobamovirus CP with the identical specificity as reported for N'. Analysis of N'-containing and tobamovirus-susceptible N. tabacum accessions supported the hypothesis that the candidate is the N' gene itself. Chimera analysis between N' and L(3) revealed that their LRR domains determine the spectrum of their tobamovirus CP recognition. Deletion and mutation analyses of N' and L(3) revealed that the conserved sequences in their C-terminal regions have important roles but contribute differentially to the recognition of common avirulence proteins. The results collectively suggest that Nicotiana N' and Capsicum L genes, which most likely evolved from a common ancestor, differentiated in their recognition specificity through changes in the structural requirements for LRR function.

Large-scale gene disruption in Magnaporthe oryzae identifies MC69, a secreted protein required for infection by monocot and dicot fungal pathogens.

To search for virulence effector genes of the rice blast fungus, Magnaporthe oryzae, we carried out a large-scale targeted disruption of genes for 78 putative secreted proteins that are expressed during the early stages of infection of M. oryzae. Disruption of the majority of genes did not affect growth, conidiation, or pathogenicity of M. oryzae. One exception was the gene MC69. The mc69 mutant showed a severe reduction in blast symptoms on rice and barley, indicating the importance of MC69 for pathogenicity of M. oryzae. The mc69 mutant did not exhibit changes in saprophytic growth and conidiation. Microscopic analysis of infection behavior in the mc69 mutant revealed that MC69 is dispensable for appressorium formation. However, mc69 mutant failed to develop invasive hyphae after appressorium formation in rice leaf sheath, indicating a critical role of MC69 in interaction with host plants. MC69 encodes a hypothetical 54 amino acids protein with a signal peptide. Live-cell imaging suggested that fluorescently labeled MC69 was not translocated into rice cytoplasm. Site-directed mutagenesis of two conserved cysteine residues (Cys36 and Cys46) in the mature MC69 impaired function of MC69 without affecting its secretion, suggesting the importance of the disulfide bond in MC69 pathogenicity function. Furthermore, deletion of the MC69 orthologous gene reduced pathogenicity of the cucumber anthracnose fungus Colletotrichum orbiculare on both cucumber and Nicotiana benthamiana leaves. We conclude that MC69 is a secreted pathogenicity protein commonly required for infection of two different plant pathogenic fungi, M. oryzae and C. orbiculare pathogenic on monocot and dicot plants, respectively.

Serine palmitoyltransferase, the first step enzyme in sphingolipid biosynthesis, is involved in nonhost resistance.

An overexpression screen of Nicotiana benthamiana cDNAs identified a gene for the LCB2 subunit of serine palmitoyltransferase (SPT) as a potent inducer of hypersensitive response-like cell death. The pyridoxal 5'-phosphate binding site of NbLCB2 is required for its function as a cell death inducer. NbLCB2 mRNA is accumulated after infection by nonhost pathogen Pseudomonas cichorii. Resistance of N. benthamiana against P. cichorii was compromised by treatment with an SPT inhibitor and in NbLCB2- and NbLCB1-silenced plants. These results suggest that biosynthesis of sphingolipids is necessary for the nonhost resistance of N. benthamiana against P. cichorii.

High-throughput in planta expression screening identifies an ADP-ribosylation factor (ARF1) involved in non-host resistance and R gene-mediated resistance.

To identify positive regulators of cell death in plants, we performed a high-throughput screening, employing potato virus X-based overexpression in planta of a cDNA library derived from paraquat-treated Nicotiana benthamiana leaves. The screening of 30,000 cDNA clones enabled the identification of an ADP-ribosylation factor 1 (ARF1) that induces cell death when overexpressed in N. benthamiana. Overexpression of the guanosine diphosphate (GDP)-locked mutant of ARF1 did not trigger cell death, suggesting that ARF1 guanosine triphosphatase (GTPase) activity is necessary for the observed cell death-inducing activity. The ARF1 transcript level increased strongly following treatment with Phytophthora infestans elicitor INF1, as well as inoculation with a non-host pathogen Pseudomonas cichorii in N. benthamiana. In addition, ARF1 was induced in the interaction between the N gene and tobacco mosaic virus (TMV) in Nicotiana tabacum. By contrast, inoculation with the virulent pathogen Pseudomonas syringae pv. tabaci did not affect ARF1 expression in N. benthamiana. Virus-induced gene silencing of ARF1 in N. benthamiana resulted in a stunted phenotype, and severely hampered non-host resistance towards P. cichorii. In addition, ARF1 silencing partially compromised resistance towards TMV in N. benthamiana containing the N resistance gene. By contrast, and in accordance with the ARF1 gene expression profile, silencing of ARF1 transcription did not alter the susceptibility of N. benthamiana towards the pathogen P. syringae pv. tabaci. These results strongly implicate ARF1 in the non-host resistance to bacteria and N gene-mediated resistance in N. benthamiana.

NbLRK1, a lectin-like receptor kinase protein of Nicotiana benthamiana, interacts with Phytophthora infestans INF1 elicitin and mediates INF1-induced cell death.

Phytophthora infestans INF1 elicitin causes the hypersensitive response (HR) in Nicotiana benthamiana (Kamoun et al. in Plant Cell 10:1413-1425, 1998). To identify N. benthamiana proteins that interact with INF1, we carried out a yeast two-hybrid screen. This screen resulted in the isolation of a gene NbLRK1 coding for a novel lectin-like receptor kinase. NbLRK1 interacted with INF1 through its VIb kinase subdomain. Purified INF1 and NbLRK1 proteins also interacted in vitro. INF1 treatment of N. benthamiana leaves induced autophosphorylation of NbLRK1. Most importantly, virus-induced gene silencing (VIGS) of NbLRK1 delayed INF1-mediated HR in N. benthamiana. These data suggest that NbLRK1 is a component of the N. benthamiana protein complex that recognizes INF1 elicitor and transduces the HR signal.

Virus-induced gene silencing of 14-3-3 genes abrogates dark repression of nitrate reductase activity in Nicotiana benthamiana.

In order to study the effect of repression of 14-3-3 genes on actual activity of the nitrate reductase (NR) in Nicotiana benthamiana leaves, Nb14-3-3a gene was silenced by virus-induced gene silencing (VIGS) method using potato virus X (PVX). Expression of Nb14-3-3a as well as Nb14-3-3b genes was altogether repressed in the leaves of PVX-14-3a-infected plants. Furthermore, two-dimensional gel electrophoresis and immunoblot analysis with anti-14-3-3 antiserum suggested that the expressions of Nb14-3-3a and Nb14-3-3b proteins are accordingly repressed in PVX-14-3a-infected plants. It is well known that binding of 14-3-3 proteins to phosphorylated NR leads to substantial decrease in NR activity of leaves under darkness. Therefore, we studied the changes in NR activity in response to light/dark transitions in the leaves of PVX-14-3a-infected plants. NR activation state was kept at a high level under darkness in PVX-14-3a-infected plants, but not in PVX-green fluorescent protein (GFP)-infected and control plants. This result suggests that Nb14-3-3a and/or Nb14-3-3b proteins are indeed involved in the inactivation of NR activity under darkness in N. benthamiana.

A high-throughput screen of cell-death-inducing factors in Nicotiana benthamiana identifies a novel MAPKK that mediates INF1-induced cell death signaling and non-host resistance to Pseudomonas cichorii.

A high-throughput overexpression screen of Nicotiana benthamiana cDNAs identified a gene for a mitogen-activated protein kinase kinase (MAPKK) as a potent inducer of the hypersensitive response (HR)-like cell death. NbMKK1 protein is localized to the nucleus, and the N-terminal putative MAPK docking site of NbMKK1 is required for its function as a cell-death inducer. NbMKK1-mediated leaf-cell death was compromised in leaves where NbSIPK expression was silenced by virus-induced gene silencing. A yeast two-hybrid assay showed that NbMKK1 and NbSIPK physically interact, suggesting that NbSIPK is one of the downstream targets of NbMKK1. Phytophthora infestans INF1 elicitor-mediated HR was delayed in NbMKK1-silenced plants, indicating that NbMKK1 is involved in this HR pathway. Furthermore, the resistance of N. benthamiana to a non-host pathogen Pseudomonas cichorii was compromised in NbMKK1-silenced plants. These results demonstrate that MAPK cascades involving NbMKK1 control non-host resistance including HR cell death.

A Novel MAPKK Involved in Cell Death and Defense Signaling.

A high-throughput in planta overexpression screen of a Nicotiana benthamiana cDNA library identified a mitogen activated protein kinase kinase (MAPKK), NbMKK1, as a potent inducer of hypersensitive response (HR)-like cell death. NbMKK1-mediated cell death was attenuated in plants whereby expression of NbSIPK, an ortholog of tobacco SIPK and Arabidopsis AtMPK6, was knocked down by virus-induced gene silencing (VIGS), suggesting that NbMKK1 functions upstream of NbSIPK. In accordance with this result, NbMKK1 phosphorylated NbSIPK in vitro, and furthermore NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. VIGS of NbMKK1 in N. benthamiana resulted in a delay of Phytophthora infestans INF1 elicitin-mediated HR as well as in the reduction of resistance against a non-host pathogen Pseudomonas cichorii. Our data of NbMKK1, together with that of LeMKK4,1 demonstrate the presence of a novel defense signaling pathway involving NbMKK1/LeMKK4 and SIPK.

Tobacco ZFT1, a transcriptional repressor with a Cys2/His2 type zinc finger motif that functions in spermine-signaling pathway.

We previously proposed that a spermine (Spm)-mediated signal transduction pathway is involved in the hypersensitive response induced by Tobacco mosaic virus (TMV) in tobacco plants. To identify regulatory component(s) of this pathway, we surveyed a tobacco cDNA library and found that the ZFT1 gene, which encodes a Cys2/His2 type zinc-finger protein, is Spm-responsive. ZFT1 was not induced by two other polyamines, putrescine and spermidine, or by salicylic acid (SA), jasmonic acid or ethylene. Furthermore, ZFT1 was upregulated in TMV- inoculated tobacco plants in an N gene-dependent manner. Notably, induction of ZFT1 by Spm and by TMV infection was unimpaired in NahG-transgenic tobacco plants, indicating that cross-talk with an SA signaling pathway is not involved in this response. Within the Spm-signaling pathway, we found that ZFT1 functioned downstream of both mitochondrial dysfunction and mitogen-activated protein kinase activation. The ZFT1 protein has two zinc finger motifs and shows a high degree of similarity to ZPT2-3 in petunia and SCOF1 in soybean. However, unlike the latter two proteins, ZFT1 binds to the EP1S sequence and functions as a transcription repressor. Moreover, interestingly, ZFT1 overexpression rendered tobacco plants more tolerant to TMV. Based on the results presented here, we propose that ZFT1 functions as a transcription repressor in a Spm signaling pathway, thereby accelerating necrotic local region formation in tobacco leaves.

High-throughput in planta expression screening identifies a class II ethylene-responsive element binding factor-like protein that regulates plant cell death and non-host resistance.

We performed high-throughput screening using the potato virus X (PVX) system to overexpress Nicotiana benthamiana genes in planta and identify positive regulators of cell death. This screening identified NbCD1, a novel class II ethylene-responsive element binding factor (ERF), as a potent inducer of the hypersensitive response (HR)-like cell death. NbCD1 expression was induced by treatments with INF1 elicitor and a non-host pathogen Pseudomonas cichorii. NbCD1 exhibited transcriptional repressor activity through its EAR motif, and this motif was necessary for NbCD1 to cause cell death. We identified 58 genes that displayed altered transcription following NbCD1 overexpression. NbCD1 overexpression downregulated the expression of HSR203, a negative regulator of hypersensitive death. Conditional expression of NbCD1 in Arabidopsis also caused cell death, indicating that NbCD1 downstream cascades are conserved in dicot plants. To further confirm the role of NbCD1 in defense, we used virus-induced gene silencing to demonstrate that NbCD1 is required for non-host resistance of N. benthamiana to the bacterial pathogen P. cichorii. Our data point to a model of transcriptional regulatory cascades. NbCD1 positively regulates cell death and contributes to non-host resistance, possibly by downregulating the expression of other defense response genes.

Ethylene-mediated cross-talk between calcium-dependent protein kinase and MAPK signaling controls stress responses in plants.

Plants are constantly exposed to environmental changes and need to integrate multiple external stress cues. Calcium-dependent protein kinases (CDPKs) are implicated as major primary Ca2+ sensors in plants. CDPK activation, like activation of mitogen-activated protein kinases (MAPKs), is triggered by biotic and abiotic stresses, although distinct stimulus-specific stress responses are induced. To investigate whether CDPKs are part of an underlying mechanism to guarantee response specificity, we identified CDPK-controlled signaling pathways. A truncated form of Nicotiana tabacum CDPK2 lacking its regulatory autoinhibitor and calcium-binding domains was ectopically expressed in Nicotiana benthamiana. Infiltrated leaves responded to an abiotic stress stimulus with the activation of biotic stress reactions. These responses included synthesis of reactive oxygen species, defense gene induction, and SGT1-dependent cell death. Furthermore, N-terminal CDPK2 signaling triggered enhanced levels of the phytohormones jasmonic acid, 12-oxo-phytodienoic acid, and ethylene but not salicylic acid. These responses, commonly only observed after challenge with a strong biotic stimulus, were prevented when the CDPK's intrinsic autoinhibitory peptide was coexpressed. Remarkably, elevated CDPK signaling compromised stress-induced MAPK activation, and this inhibition required ethylene synthesis and perception. These data indicate that CDPK and MAPK pathways do not function independently and that a concerted activation of both pathways controls response specificity to biotic and abiotic stress.

A subset of hypersensitive response marker genes, including HSR203J, is the downstream target of a spermine signal transduction pathway in tobacco.

A cellular signal transduction pathway induced by the polyamine, spermine (Spm), and transmitted by mitochondrial dysfunction is proposed in tobacco. In this investigation, we further resolve the pathway by identifying a subset of hypersensitive response (HR) marker genes as downstream components. In a previous report, we identified harpin-induced 1 (HIN1) and two closely related genes as responsive to Spm. Other HR marker genes, HSR203J, HMGR, HSR201, and HSR515, are also Spm-responsive. Induction of these HR marker genes, including HIN1, by Spm was suppressed by pre-treatment with antioxidants, calcium channel blockers, inhibitor of mitochondrial permeability transition pore openings, and blockers of amine oxidase/polyamine oxidase. Such quenching is also observed for Spm-induced activation of two mitogen-activated protein kinases (MAPKs), salicylic acid-induced protein kinase (SIPK), and wound-induced protein kinase (WIPK), and upregulation of the WIPK gene, suggesting that all these components are part of the same signaling pathway. Furthermore, gain-of-function and loss-of-function studies on MAPK cascade members reveal that the expression of Spm-induced HR marker genes varies with respect to involvement of SIPK/WIPK activation.

Gene expression analysis of plant host-pathogen interactions by SuperSAGE.

The type III restriction endonuclease EcoP15I was used in isolating fragments of 26 bp from defined positions of cDNAs. We call this substantially improved variant to the conventional serial analysis of gene expression (SAGE) procedure "SuperSAGE." By applying SuperSAGE to Magnaporthe grisea (blast)-infected rice leaves, gene expression profiles of both the rice host and blast fungus were simultaneously monitored by making use of the fully sequenced genomes of both organisms, revealing that the hydrophobin gene is the most actively transcribed M. grisea gene in blast-infected rice leaves. Moreover, SuperSAGE was applied to study gene expression changes before the so-called hypersensitive response in INF1 elicitor-treated Nicotiana benthamiana, a "nonmodel" organism for which no DNA database is available. Again, SuperSAGE allowed rapid identification of genes up- or down-regulated by the elicitor. Surprisingly, many of the down-regulated genes coded for proteins involved in photosynthesis. SuperSAGE will be especially useful for transcriptome profiling of two or more interacting organisms like hosts and pathogens, and of organisms, for which no DNA database is available.

C-terminal domain of a hevein-like protein from Wasabia japonica has potent antimicrobial activity.

An antimicrobial protein, designated WjAMP-1, was purified from leaves of Wasabia japonica L. WjAMP-1 showed antimicrobial activity against both fungi and bacteria. The deduced amino acid sequence of cDNA of WjAMP-1 showed 60% and 70% identity with a hevein from Hevea brasiliansis and a hevein-like protein from Arabidopsis thaliana, respectively. However, matured WjAMP-1 lacked the hevein domain and may correspond to the C-terminal domain of hevein. Southern blot analysis showed that one or two copies of the WjAMP-1 gene were presented in the genome of wasabi. Expression of WjAMP-1 was detected in all organs tested, and was especially strong in petioles. Expression of WjAMP-1 was induced by the inoculation with fungal pathogens and treatment with methyl jasmonate. Recombinant WjAMP-1 expressed in Nicotiana benthamiana using potato virus X vector also inhibited not only growth of fungi but also bacteria. These results suggest that WjAMP-1 may be the C-terminal domain of hevein and one of the defense gene in W. japonica. WjAMP-1 gene may be useful genes to generate resistant plants against fungal and bacterial pathogens.

Virus-induced silencing of FtsH gene in Nicotiana benthmiana causes a striking bleached leaf phenotype.

A recombinant Potato virus X (PVX) vector, pTXS.FtsH, harboring partial sequence of FtsH gene of Nicotiana benthamiana was constructed to silence the expression of endogenous FtsH homologous gene in N. benthamiana. Inoculation with in vitro runoff transcript of pTXS.FtsH to N. benthamiana plants allowed silencing of FtsH, causing striking bleaching of upper leaves reminiscent of var2 mutant phenotype of Arabidopsis thaliana. FtsH-silenced plants exhibited no resistance against Tobacco mosaic virus (TMV) and a phytopathogenic fungus Botrytis cinerea. Virus-induced gene silencing (VIGS) of N. benthamiana with PVX as demonstrated here would be an efficient and rapid method to study the function of other elements of photosystem II (PSII) in planta.