Plants have a sensitive system that detects various pathogenderived molecules to protect against infection. Flagellin, a main component of the bacterial flagellum, from the rice avirulent N1141 strain of the Gram-negative phytopathogenic bacterium Acidovorax avenae induces plant immune responses including H 2 O 2 generation, whereas flagellin from the rice virulent K1 strain of A. avenae does not induce these immune responses. To clarify the molecular mechanism that leads to these differing responses between the K1 and N1141 flagellins, recombinant K1 and N1141 flagellins were generated using an Escherichia coli expression system. During development, plants are continuously confronted with diverse pathogens. However, plants are resistant to most microbes and rely entirely on plant immune responses for their defense. Plants have evolved a multilayered defense system that can be activated upon pathogen invasion. The first layer recognizes conserved microbial molecules, referred to as microbeassociated molecular patterns, via pattern recognition receptors (1, 2). Microbe-associated molecular pattern-triggered immunity is key to plant innate immunity (3). Successful pathogens can deliver effectors that suppress these immune responses and contribute to pathogen virulence (4). Another layer recognizes pathogen effector molecules through host resistance genes, triggering a rapid defense response that often includes a localized programmed cell death reaction known as the hypersensitive response (5-7).Microbe-associated molecular patterns include structures characteristic of pathogens, such as ␤-glucan, polysaccharide chitin, ergosterol, lipopolysaccharides (LPS), flagellin, and elongation factor Tu (8 -13). Among these microbe-associated molecular patterns, flagellin, a main component of the bacterial flagellum, has been the most extensively studied in regard to the recognition mechanism and signal transduction. Arabidopsis recognizes the most conserved N-terminal domain of flagellin that consists of a 22-amino acid peptide (flg22) 2 (12). Recognition of this elicitor-active domain depends on flagellin sensing 2 (FLS2) (14). FLS2 encodes a receptor-like kinase composed of an extracellular leucine-rich repeat, a single membrane-spanning domain, and a cytoplasmic serine/threonine kinase domain. FLS2 and flg22 were shown to physically interact by chemical cross-linking and immunoprecipitation studies, suggesting that FLS2 determines the specificity in recognizing flagellin (15).Acidovorax avenae is a Gram-negative bacterium that causes a seedling disease that is characterized by the formation of brown stripes on the sheaths of infected plants. A. avenae has a wide host range among monocotyledonous plants; however, individual strains of this pathogen infect only one or a few host species (16). For example, strains isolated from rice, such as K1 and H8301, can infect only rice plants (virulent), whereas the N1141 strain isolated from finger millet cannot infect rice even after it is inoculated into rice tissues (avirulent). ...

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Microbial pathogens deliver effectors into plant cells to suppress plant immune responses and modulate host metabolism in order to support infection processes. We sought to determine if the Acidovorax avenae rice-virulent K1 strain can suppress pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) induced by flagellin isolated from the rice-avirulent N1141 strain. The flagellin-triggered PTI, including H2O2 generation, callose deposition, and expression of several immune-related genes were strongly suppressed in K1 pre-inoculated cultured rice cells in a Type III secretion system (T3SS)-dependent manner. By screening 4,562 transposon-tagged mutants based on their suppression ability, we found that 156 transposon-tagged K1 mutants lost the ability to suppress PTI induction. Mutant sequence analysis, comprehensive expression analysis using RNA-sequencing, and the prediction of secretion through T3SS showed that a protein named A. avenae K1 suppression factor 1 (AKSF1) suppresses flagellin-triggered PTI in rice. Translocation of AKSF1 protein into rice cells is dependent on T3SS during infection, an AKSF1-disruption mutant lost the ability to suppress PTI responses, and expression of AKSF1 in AKSF1-disruption mutant complemented the suppression activity. When AKSF1-disruption mutants were inoculated into the host rice plant, reduction of the disease symptoms and suppression of the bacterial growth were observed. Taken together, our results demonstrate that AKSF1 is a novel effector that can suppress the PTI in host rice plant.

The first layer of active plant immunity relies upon the recognition of PAMPs or MAMPs, and the induction of PTI. Flagellin is the major protein component of the bacterial flagellum. Flagellin-derived peptide fragments such as CD2–1, flg22, and flgII-28 function as PAMPs in most higher plants. To determine the distribution of CD2–1, flg22, and flgII-28 recognition systems within plant species, the inducibility of PTI by CD2–1, flg22, and flgII-28 in eight plant species, including monocotyledonous and dicotyledonous plants was investigated. CD2–1 caused PTI responses in Oryza sativa, Brachypodium distachyon, and Asparagus persicus; flg22 caused PTI responses in Phyllostachys nigra, A. persicus, Arabidopsis thaliana, Nicotiana tabacum, Solanum lycopersicum, and Lotus japonicus; and flgII-28 caused PTI responses only in S. lycopersicum. Furthermore, quantitative analysis of FLS2 receptor revealed that the responsiveness of flg22 in plants was dependent on the expression level of the receptor.

Many plant pathogens inject type III (T3SS) effectors into host cells to suppress host immunity and promote successful infection. The bacterial pathogen Acidovorax avenae causes brown stripe symptom in many species of monocotyledonous plants; however, individual strains of each pathogen infect only one host species. T3SS-deleted mutants of A. avenae K1 (virulent to rice) or N1141 (virulent to finger millet) caused no symptom in each host plant, suggesting that T3SS effectors are involved in the symptom formation. To identify T3SS effectors as virulence factors, we performed whole-genome and predictive analyses. Although the nucleotide sequence of the novel leucine-rich repeat protein (Lrp) gene of N1141 had high sequence identity with K1 Lrp, the amino acid sequences of the encoded proteins were quite different due to a 1-bp insertion within the K1 Lrp gene. An Lrp-deleted K1 strain (KΔLrp) did not cause brown stripe symptom in rice (host plant for K1); by contrast, the analogous mutation in N1141 (NΔLrp) did not interfere with infection of finger millet. In addition, NΔLrp retained the ability to induce effector-triggered immunity (ETI), including hypersensitive response cell death and expression of ETI-related genes. These data indicated that K1 Lrp functions as a virulence factor in rice, whereas N1141 Lrp does not play a similar role in finger millet. Yeast two-hybrid screening revealed that K1 Lrp interacts with oryzain α, a pathogenesis-related protein of the cysteine protease family, whereas N1141 Lrp, which contains LRR domains, does not. This specific interaction between K1 Lrp and oryzain α was confirmed by Bimolecular fluorescence complementation assay in rice cells. Thus, K1 Lrp protein may have acquired its function as virulence factor in rice due to a frameshift mutation.

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PolyADP-ribosylation plays an essential function in maintenance of genomic stability and cell survival. Although there are several proteins served as acceptor proteins in vitro, there are few proteins in vivo that are identified, including poly(ADP-ribose) polymerase-1. We have been studying to analyze the mechanism of neuronal cell death observed in poly(ADP-ribose) glycohydrolase (PARG)-knockout Drosophila melanogaster that shows accumulation of polyADP-ribosylated proteins in the brain. As the first step, we have been trying to isolate the polyADP-ribosylated accepter proteins from the PARG-knockout fly. The strategy is to extract the polyADP-ribosylated proteins and isolate them with affinity chromatography using monoclonal antibody against poly(ADP-ribose) (PAR) (10H). The bound fraction was eluted by buffer containing salt. Next, part of eluted fraction is treated with NaOH for separating the proteins from PAR chain. Nontreated fraction and treated fraction were separated with two-dimensional gel electrophoresis. After protein staining, the specific spots that were newly found after NaOH treatment were candidate acceptor proteins for polyADP-ribosylation in vivo and could be analyzed with liquid chromatography-mass spectrometry. We present the procedure to this approach.

Plant pathogenic bacteria inject effectors into plant cells using type III secretion systems (T3SS) to evade plant immune systems and facilitate infection. In contrast, plants have evolved defense systems called effector-triggered immunity (ETI) that can detect such effectors during co-evolution with pathogens. The rice-avirulent strain N1141 of the bacterial pathogen Acidovorax avenae causes rice ETI, including hypersensitive response (HR) cell death in a T3SS-dependent manner, suggesting that strain N1141 expresses an ETI-inducing effector. By screening 6,200 transposon-tagged N1141 mutants based on their ability to induce HR cell death, we identified 17 mutants lacking this ability. Sequence analysis and T3SS-mediated intracellular transport showed that a protein called rice HR cell death inducing factor (RHIF) is a candidate effector protein that causes HR cell death in rice. RHIF-disrupted N1141 lacks the ability to induce HR cell death, whereas RHIF expression in this mutant complemented this ability. In contrast, RHIF from rice-virulent strain K1 functions as an ETI inducer in the non-host plant finger millet. Furthermore, inoculation of rice and finger millet with either RHIF-deficient N1141 or K1 strains showed that a deficiency of RHIF genes in both strains results in decreased infectivity toward each the host plants. Collectively, novel effector RHIFs identified from A. avenae strains N1141 and K1 function in establishing infection in host plants and in ETI induction in non-host plants.