A Seed-Borne Bacterium of Rice, Pantoea dispersa BB1, Protects Rice from the Seedling Rot Caused by the Bacterial Pathogen Burkholderia glumae.

Seedling rot, caused by the bacterial pathogen Burkholderia glumae, is a major disease of rice. It originates from pathogen-contaminated seeds and is thus mainly controlled by pesticide treatments of seeds. We previously demonstrated that the seed-borne bacteria of rice may be a useful and sustainable alternative to pesticides to manage seedling rot, but they are limited in terms of variety. Here, we report that another seed-borne bacterium, Pantoea dispersa BB1, protects rice from B. glumae. We screened 72 bacterial isolates from rice seeds of three genetically different cultivars inoculated or non-inoculated with B. glumae. 16S rRNA gene sequencing revealed that pathogen inoculation affected the composition of culturable seed-borne bacterial communities and increased the presence of Pantoea and Paenibacillus species. Among three Pantoea and Paenibacillus isolates that exhibit tolerance to toxoflavin, a virulence factor of B. glumae, P. dispersa BB1 significantly mitigated the symptoms of rice seedling rot. The culture filtrate of BB1 inhibited the growth of B. glumae in vitro, suggesting that this isolate secretes antibacterial compounds. Seed treatment with BB1 suppressed pathogen propagation in plants, although seed treatment with the culture filtrate did not. Because BB1 did not show pathogenicity in rice, our findings demonstrate that BB1 is a promising biocontrol agent against seedling rot.

Multiple endogenous seed-born bacteria recovered rice growth disruption caused by Burkholderia glumae.

Burkholderia glumae is a causal agent of bacterial grain and seedling rot in rice, and is a threat to stable global food supply. The virulence of B. glumae was suppressed when it was inoculated on budding seed rather than on non-budding seed. To clarify the phenomena, pathogen titer inside the rice plant was measured by serial dilution plating of lysates from budding rice seedlings. Surprisingly, morphologically different types of colonies were observed on the plates. These 'contaminated' rice seed-born bacteria (RSB) were identified by sequencing 16S rRNA genes as three strains of Pseudomonas putida (RSB1, RSB10, RSB15) and Stenotrophomonas maltophilia (RSB2). All bacteria and B. glumae were simultaneously inoculated onto rice seeds, and all three P. putida RSBs suppressed the growth disruption caused by B. glumae, whereas RSB2 had no effect. Thus, the virulence was synergistically suppressed when co-treated with RSBs. The effect could be dependent on the high biofilm formation ability of RSB2. By comprehensive microbiota analysis, endogenous rice flora were changed by RSBs treatment. These results suggest the possibility of novel pathogen control through pre-treatment with endogenous beneficial microorganisms. The method would contribute substantially to the implementation of sustainable agriculture stated in Sustainable Development Goals of United Nations.

Loss of chloroplast-localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection.

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine-specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62- and atpp2c26-deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis-related protein, chaperonin-60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two-dimensional isoelectric focusing sodium dodecylsulfate-polyacrylamide gel electrophoresis (2D-IDF-SDS-PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.

Real time live imaging of phytopathogenic bacteria Xanthomonas campestris pv. campestris MAFF106712 in 'plant sweet home'.

Xanthomonas is one of the most widespread phytobacteria, causing diseases on a variety of agricultural plants. To develop novel control techniques, knowledge of bacterial behavior inside plant cells is essential. Xanthomonas campestris pv. campestris, a vascular pathogen, is the causal agent of black rot on leaves of Brassicaceae, including Arabidopsis thaliana. Among the X. campestris pv. campestris stocks in the MAFF collection, we selected XccMAFF106712 as a model compatible pathogen for the A. thaliana reference ecotype Columbia (Col-0). Using modified green fluorescent protein (AcGFP) as a reporter, we observed real time XccMAFF106712 colonization in planta with confocal microscopy. AcGFP-expressing bacteria colonized the inside of epidermal cells and the apoplast, as well as the xylem vessels of the vasculature. In the case of the type III mutant, bacteria colonization was never detected in the xylem vessel or apoplast, though they freely enter the xylem vessel through the wound. After 9 days post inoculation with XccMAFF106712, the xylem vessel became filled with bacterial aggregates. This suggests that Xcc colonization can be divided into main four steps, (1) movement in the xylem vessel, (2) movement to the next cell, (3) adhesion to the host plant cells, and (4) formation of bacterial aggregates. The type III mutant abolished at least steps (1) and (2). Better understanding of Xcc colonization is essential for development of novel control techniques for black rot.

XopR, a type III effector secreted by Xanthomonas oryzae pv. oryzae, suppresses microbe-associated molecular pattern-triggered immunity in Arabidopsis thaliana.

Xanthomonas oryzae pv. oryzae is the causal agent of bacterial blight of rice. The XopR protein, secreted into plant cells through the type III secretion apparatus, is widely conserved in xanthomonads and is predicted to play important roles in bacterial pathogenicity. Here, we examined the function of XopR by constructing transgenic Arabidopsis thaliana plants expressing it under control of the dexamethasone (DEX)-inducible promoter. In the transgenic plants treated with DEX, slightly delayed growth and variegation on leaves were observed. Induction of four microbe-associated molecular pattern (MAMP)-specific early-defense genes by a nonpathogenic X. campestris pv. campestris hrcC deletion mutant were strongly suppressed in the XopR-expressing plants. XopR expression also reduced the deposition of callose, an immune response induced by flg22. When transiently expressed in Nicotiana benthamiana, a XopR::Citrine fusion gene product localized to the plasma membrane. The deletion of XopR in X. oryzae pv. oryzae resulted in reduced pathogenicity on host rice plants. Collectively, these results suggest that XopR inhibits basal defense responses in plants rapidly after MAMP recognition.

Identification and characterization of 2'-deoxyuridine from the supernatant of conidial suspensions of rice blast fungus as an infection-promoting factor in rice plants.

We previously detected infection-promoting activity in the supernatant of the conidial suspension (SCS) of the rice blast fungus. In the present study, a molecule carrying the activity was purified and identified as 2'-deoxyuridine (dU). The infection-promoting activity of dU was strictly dependent on its chemical structure and displayed characteristics consistent with those of the SCS. Notably, the activity of dU was exclusively detected during interactions between rice and virulent isolates of the fungus, the number of susceptible lesions in leaf blades was increased by dU, and nonhost resistance in rice plants was not affected by treatment with dU. In addition, the expression of pathogensis-related genes, accumulation of H(2)O(2), and production of phytoalexins in rice in response to inoculation with virulent fungal isolates was not suppressed by dU. The infection-promoting activity of dU was not accompanied by elevated levels of endogenous abscissic acid, which is known to modify plant-pathogen interactions, and was not detected in interactions between oat plants and a virulent oat blast fungus isolate. Taken together, these results demonstrate that dU is a novel infection-promoting factor that acts specifically during compatible interactions between rice plants and rice blast fungus in a mode distinct from that of toxins and suppressors.

The Pseudomonas syringae type III effector HopAM1 enhances virulence on water-stressed plants.

Pseudomonas syringae strains deliver diverse type III effector proteins into host cells, where they can act as virulence factors. Although the functions of the majority of type III effectors are unknown, several have been shown to interfere with plant basal defense mechanisms. Type III effectors also could contribute to bacterial virulence by enhancing nutrient uptake and pathogen adaptation to the environment of the host plant. We demonstrate that the type III effector HopAM1 (formerly known as AvrPpiB) enhances the virulence of a weak pathogen in plants that are grown under drought stress. This is the first report of a type III effector that aids pathogen adaptation to water availability in the host plant. Expression of HopAM1 makes transgenic Ws-0 Arabidopsis hypersensitive to abscisic acid (ABA) for stomatal closure and germination arrest. Conditional expression of HopAM1 in Arabidopsis also suppresses basal defenses. ABA responses overlap with defense responses and ABA has been shown to suppress defense against P. syringae pathogens. We propose that HopAM1 aids P. syringae virulence by manipulation of ABA responses that suppress defense responses. In addition, host ABA responses enhanced by type III delivery of HopAM1 protect developing bacterial colonies inside leaves from osmotic stress.

Characterization of an elicitor-induced rice WRKY gene, OsWRKY71.

Expression of OsWRKY71, a rice WRKY gene, was induced by biotic elicitors and pathogen infection. It was also found that OsWRKY71 has features characteristic of a transcriptional repressor. Microarray analysis revealed that several elicitor-induced defense-related genes were upregulated in rice cells overexpressing OsWRKY71. These results indicate that the activation of defense-related genes by OsWRKY71 was probably indirect.

Involvement of the elicitor-induced gene OsWRKY53 in the expression of defense-related genes in rice.

We present a detailed characterization of the chitin oligosaccharide elicitor-induced gene OsWRKY53. OsWRKY53 was also induced in suspension-cultured rice cells by a fungal cerebroside elicitor and in rice plants by infection with the blast fungus Magnaporthe grisea. A fusion of OsWRKY53 with green fluorescent protein was detected exclusively in the nuclei of onion epidermal cells, and OsWRKY53 protein specifically bound to W-box elements. A transient assay using the particle bombardment method showed that OsWRKY53 is a transcriptional activator. A microarray analysis revealed that several defense-related genes, including pathogenesis-related protein genes such as PBZ1, were upregulated in rice cells overexpressing OsWRKY53. Finally, overexpression of OsWRKY53 in rice plants resulted in enhanced resistance to M. grisea. These results strongly suggest that OsWRKY53 is a transcription factor that plays important roles in elicitor-induced defense signaling pathways in rice.

Preparation of mixed alginate elicitors with high activity for the efficient production of 5'-phosphodiesterase by Catharanthus roseus cells.

When various autoclaved microbial cells suspensions (exogenous elicitors) were added to Catharanthus roseus cell cultures, its growth was inhibited but 5'-phosphodiesterase (PDase) production was stimulated. The greatest effect was with autoclaved Alteromonas macleodii: the dry cell concentration decreased from 13 to 10.9 mg/ml while PDase production increased from 0.022 to 0.235 U/ml. A combination of A. macleodii (as exogenous elicitor) and 0.1%(w/v) alginate oligomers (AO: acting as both endogenous elicitor and scavenger of active oxygen species) minimized the cell growth inhibition but enhanced PDase production (0.474 U/ml) about 20 times higher than the control (no addition). The method for the preparation of mixed alginate elicitors with high activities containing exogenous elicitor (autoclaved A. macleodii), endogenous elicitor (AO), and trans-4,5-dihydroxy-2-cyclopenten-1-one was developed. The mixed alginate elicitors significantly promoted PDase production (2.67 U/ml) by C. roseus, and the productivity was increased 120-fold compared to the control without cell growth inhibition.

Plant cells recognize chitin fragments for defense signaling through a plasma membrane receptor.

Chitin is a major component of fungal cell walls and serves as a molecular pattern for the recognition of potential pathogens in the innate immune systems of both plants and animals. In plants, chitin oligosaccharides have been known to induce various defense responses in a wide range of plant cells including both monocots and dicots. To clarify the molecular machinery involved in the perception and transduction of chitin oligosaccharide elicitor, a high-affinity binding protein for this elicitor was isolated from the plasma membrane of suspension-cultured rice cells. Characterization of the purified protein, CEBiP, as well as the cloning of the corresponding gene revealed that CEBiP is actually a glycoprotein consisting of 328 amino acid residues and glycan chains. CEBiP was predicted to have a short membrane spanning domain at the C terminus. Knockdown of CEBiP gene by RNA interference resulted in the suppression of the elicitor-induced oxidative burst as well as the gene responses, showing that CEBiP plays a key role in the perception and transduction of chitin oligosaccharide elicitor in the rice cells. Structural analysis of CEBiP also indicated the presence of two LysM motifs in the extracellular portion of CEBiP. As the LysM motif has been known to exist in the putative Nod-factor receptor kinases involved in the symbiotic signaling between leguminous plants and rhizobial bacteria, the result indicates the involvement of partially homologous plasma membrane proteins both in defense and symbiotic signaling in plant cells.

Rice gene expression in response to N-acetylchitooligosaccharide elicitor: comprehensive analysis by DNA microarray with randomly selected ESTs.

N-acetylchitooligosaccharides are potent elicitors to suspension-cultured rice cells, inducing a set of defense reactions. Expression of defense-related genes is considered to play an important role in defense reactions, and we employed microarray analysis of 8987 randomly selected expressed sequence tags to analyze the changes in gene expression caused by N-acetylchitooctaose. In this experiment, 166 genes were significantly induced and 93 genes were repressed. RNA gel blot analysis of 16 of these genes confirmed the microarray results. Of the 259 ESTs identified as responsive to N-acetylchytooctaose, 18 genes are related to signal transduction, including five calcium-dependent protein kinases (CDPKs). Among these, three novel CDPKs responsive to N-acetylchitooctaose were isolated.

Production of 5'-phosphodiesterase by Catharanthus roseus cells promoted by heat-degraded products generated from uronic acid.

Polyalginate was autoclaved at 121 degrees C for 20 min and its molecular weight distribution was analyzed. The autoclaved alginate yielded alginate polymer, oligomer and heat degraded products (HDPs). Each of the separated substances promoted 5'-phosphodiesterase (5'-PDase) production in suspension culture of Catharanthus roseus cells. HDPs could also be generated from other uronic acids (galacturonic acid and glucuronic acid) by autoclave treatment. The most effective substance in the HDPs was isolated and characterized as trans-4,5-dihydroxy-2-cyclopenten-1-one (DHCP). The optimal conditions for DHCP production were also established (autoclaving 1 mg/ml monogalacturonic acid [pH 2] at 121 degrees C for 2 h). A combination of oligo-alginate (below 4 kDa) and HDPs significantly promoted the production of 5'-PDase in C. roseus. Based on the above results, a novel alginate complex that gave a 44-fold increase in 5'-PDase production by C. roseus was developed.