Rhizoviticin is an alphaproteobacterial tailocin that mediates biocontrol of grapevine crown gall disease.

Tailocins are headless phage tail structures that mediate interbacterial antagonism. Although the prototypical tailocins, R- and F-pyocins, in Pseudomonas aeruginosa, and other predominantly R-type tailocins have been studied, their presence in Alphaproteobacteria remains unexplored. Here, we report the first alphaproteobacterial F-type tailocin, named rhizoviticin, as a determinant of the biocontrol activity of Allorhizobium vitis VAR03-1 against crown gall. Rhizoviticin is encoded by a chimeric prophage genome, one providing transcriptional regulators and the other contributing to tail formation and cell lysis, but lacking head formation genes. The rhizoviticin genome retains a nearly intact early phage region containing an integrase remnant and replication-related genes critical for downstream gene transcription, suggesting an ongoing transition of this locus from a prophage to a tailocin-coding region. Rhizoviticin is responsible for the most antagonistic activity in VAR03-1 culture supernatant against pathogenic A. vitis strain, and rhizoviticin deficiency resulted in a significant reduction in the antitumorigenic activity in planta. We identified the rhizoviticin-coding locus in eight additional A. vitis strains from diverse geographical locations, highlighting a unique survival strategy of certain Rhizobiales bacteria in the rhizosphere. These findings advance our understanding of the evolutionary dynamics of tailocins and provide a scientific foundation for employing rhizoviticin-producing strains in plant disease control.

Mitochondrial-targeting effector RsIA_CtaG/Cox11 in Rhizoctonia solani AG-1 IA has two functions: plant immunity suppression and cell death induction mediated by a rice cytochrome c oxidase subunit.

Rhizoctonia solani AG-1 IA causes a necrotrophic rice disease and is a serious threat to rice production. To date, only a few effectors have been characterized in AG-1 IA. We previously identified RsIA_CtaG/Cox11 and showed that infiltration of the recombinant protein into rice leaves caused disease-like symptoms. In the present study, we further characterized the functionality of RsIA_CtaG/Cox11. RsIA_CtaG/Cox11 is an alternative transcript of cytochrome c oxidase copper chaperone Cox11 that starts from the second AUG codon, but contains a functional secretion signal peptide. RNA interference with RsIA_CtaG/Cox11 reduced the pathogenicity of AG-1 IA towards rice and Nicotiana benthamiana without affecting its fitness or mycelial morphology. Transient expression of the RsIA_CtaG/Cox11-GFP fusion protein demonstrated the localization of RsIA_CtaG/Cox11 to mitochondria. Agro-infiltration of RsIA_CtaG/Cox11 into N. benthamiana leaves inhibited cell death by BAX and INF1. In contrast to rice, agro-infiltration of RsIA_CtaG/Cox11 did not induce cell death in N. benthamiana. However, cell death was observed when it was coinfiltrated with Os_CoxVIIa, which encodes a subunit of cytochrome c oxidase. Os_CoxVIIa appeared to interact with RsIA_CtaG/Cox11. The cell death triggered by coexpression of RsIA_CtaG/Cox11 and Os_CoxVIIa is independent of the leucine-rich repeat receptor kinases BAK1/SOBIR1 and enhanced the susceptibility of N. benthamiana to AG-1 IA. Two of the three evolutionarily conserved cysteine residues at positions 25 and 126 of RsIA_CtaG/Cox11 were essential for its immunosuppressive activity, but not for cell death induction. This report suggests that RsIA_CtaG/Cox11 appears to have a dual role in immunosuppression and cell death induction during pathogenesis.

The function of the plant cell wall in plant-microbe interactions.

The plant cell wall is an interface of plant-microbe interactions. The ability of microbes to decompose cell wall polysaccharides contributes to microbial pathogenicity. Plants have evolved mechanisms to prevent cell wall degradation. However, the role of the cell wall in plant-microbe interactions is not well understood. Here, we discuss four functions of the plant cell wall-physical defence, storage of antimicrobial compounds, production of cell wall-derived elicitors, and provision of carbon sources-in the context of plant-microbe interactions. In addition, we discuss the four families of cell surface receptors associated with plant cell walls (malectin-like receptor kinase family, wall-associated kinase family, leucine-rich repeat receptor-like kinase family, and lysin motif receptor-like kinase family) that have been the subject of several important studies in recent years. This review summarises the findings on both plant cell wall and plant immunity, improving our understanding and may provide impetus to various researchers.

Time-series transcriptome of Brachypodium distachyon during bacterial flagellin-induced pattern-triggered immunity.

Plants protect themselves from microorganisms by inducing pattern-triggered immunity (PTI) via recognizing microbe-associated molecular patterns (MAMPs), conserved across many microbes. Although the MAMP perception mechanism and initial events during PTI have been well-characterized, knowledge of the transcriptomic changes in plants, especially monocots, is limited during the intermediate and terminal stages of PTI. Here, we report a time-series high-resolution RNA-sequencing (RNA-seq) analysis during PTI in the leaf disks of Brachypodium distachyon. We identified 6,039 differentially expressed genes (DEGs) in leaves sampled at 0, 0.5, 1, 3, 6, and 12 hours after treatment (hat) with the bacterial flagellin peptide flg22. The k-means clustering method classified these DEGs into 10 clusters (6 upregulated and 4 downregulated). Based on the results, we selected 10 PTI marker genes in B. distachyon. Gene ontology (GO) analysis suggested a tradeoff between defense responses and photosynthesis during PTI. The data indicated the recovery of photosynthesis started at least at 12 hat. Over-representation analysis of transcription factor genes and cis-regulatory elements in DEG promoters implied the contribution of 12 WRKY transcription factors in plant defense at the early stage of PTI induction.

Secreted Glycosyltransferase RsIA_GT of Rhizoctonia solani AG-1 IA Inhibits Defense Responses in Nicotiana benthamiana.

Anastomosis group AG-1 IA of Rhizoctonia solani Khün has a wide host range and threatens crop production. Various glycosyltransferases secreted by phytopathogenic fungi play an essential role in pathogenicity. Previously, we identified a glycosyltransferase RsIA_GT (AG11A_09161) as a secreted protein-encoding gene of R. solani AG-1 IA, whose expression levels increased during infection in rice. In this study, we further characterized the virulence function of RsIA_GT. It is conserved not only in Basidiomycota, including multiple anastomosis groups of R. solani, but also in other primary fungal taxonomic categories. RsIA_GT possesses a signal peptide (SP) for protein secretion, and its functionality was proven using yeast and Nicotiana benthamiana. The SP-truncated form of RsIA_GT (RsIA_GT(ΔS)) expressed in Escherichia coli-induced lesion-like phenotype in rice leaves when applied to punched leaves. However, Agrobacterium-mediated transient expressions of both the full-length RsIA_GT and RsIA_GT(ΔS) did not induce cell death in N. benthamiana leaves. Instead, only RsIA_GT(ΔS) suppressed the cell death induced by two reference cell death factors BAX and INF1 in N.benthamiana. RsIA_GT(ΔS)R154A D168A D170A, a mutant RsIA_GT(ΔS) for the glycosyltransferase catalytic domain, still suppressed the BAX- or INF1-induced cell death, suggesting that the cell death suppression activity of RsIA_GT(ΔS) would be independent from its enzymatic activity. RsIA_GT(ΔS) also suppressed the H2O2 production and callose deposition and showed an effect on the induction of defense genes associated with the expression of BAX and INF1. The transient expression of RsIA_GT(ΔS) in N. benthamiana enhanced the lesion area caused by R. solani AG-1 IA. The secreted glycosyltransferase, RsIA_GT, of R. solani AG-1 IA is likely to have a dual role in virulence inside and outside of host cells.

HopAZ1, a type III effector of Pseudomonas amygdali pv. tabaci, induces a hypersensitive response in tobacco wildfire-resistant Nicotiana tabacum 'N509'.

Pseudomonas amygdali pv. tabaci (formerly Pseudomonas syringae pv. tabaci; Pta) is a gram-negative bacterium that causes bacterial wildfire disease in Nicotiana tabacum. The pathogen establishes infections by using a type III secretion system to inject type III effector proteins (T3Es) into cells, thereby interfering with the host__s immune system. To counteract the effectors, plants have evolved disease-resistance genes and mechanisms to induce strong resistance on effector recognition. By screening a series of Pta T3E-deficient mutants, we have identified HopAZ1 as the T3E that induces disease resistance in N. tabacum 'N509'. Inoculation with the Pta ∆hopAZ1 mutant did not induce resistance to Pta in N509. We also found that the Pta ∆hopAZ1 mutant did not induce a hypersensitive response and promoted severe disease symptoms in N509. Furthermore, a C-terminal truncated HopAZ1 abolished HopAZ1-dependent cell death in N509. These results indicate that HopAZ1 is the avirulence factor that induces resistance to Pta by N509.

Identification of Aerotaxis Receptor Proteins Involved in Host Plant Infection by Pseudomonas syringae pv. tabaci 6605.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a foliar plant pathogen that causes wildfire disease on tobacco plants. It requires chemotaxis to enter plants and establish infection. While chemotactic signals appear to be the main mechanism by which Pta6605 performs directional movement, the involvement of aerotaxis or energy taxis by this foliar pathogen is currently unknown. Based on domain structures and similarity with more than 50 previously identified putative methyl-accepting chemotaxis proteins (MCPs), the genome of Pta6605 encodes three potential aerotaxis transducers. We identified AerA as the main aerotaxis transducer and found that it possesses a taxis-to-serine-and-repellent (Tsr)-like domain structure that supports a periplasmic 4HB-type ligand-binding domain (LBD). The secondary aerotaxis transducer, AerB, possesses a cytosolic PAS-type LBD, similar to the Aer of Escherichia coli and Pseudomonas aeruginosa. Aerotaxis ability by single and double mutant strains of aerA and aerB was weaker than that by wild-type Pta6605. On the other hand, another cytosolic PAS-type LBD containing MCP did not make a major contribution to Pta6605 aerotaxis in our assay system. Furthermore, mutations in aerotaxis transducer genes did not affect surface motility or chemotactic attraction to yeast extract. Single and double mutant strains of aerA and aerB showed less colonization in the early stage of host plant infection and lower biofilm production than wild-type Pta6605. These results demonstrate the presence of aerotaxis transducers and their contribution to host plant infection by Pta6605.

Surveillance of Pathogenicity of Rhizoctonia solani Japanese Isolates with Varied Anastomosis Groups and Subgroups on Arabidopsis thaliana.

Rhizoctonia solani is a necrotrophic plant pathogen with a wide host range. R. solani is a species complex consisting of thirteen anastomosis groups (AGs) defined by compatibility of hyphal fusion reaction and subgroups based on cultural morphology. The relationship between such classifications and host specificity remains elusive. Here, we investigated the pathogenicity of seventeen R. solani isolates (AG-1 to 7) in Japan towards Arabidopsis thaliana using leaf and soil inoculations. The tested AGs, except AG-3 and AG-6, induced symptoms in both methods with variations in pathogenicity. The virulence levels differed even within the same AG and subgroup. Some isolates showed tissue-specific infection behavior. Thus, the AGs and their subgroups are suggested to be not enough to define the virulence (host and tissue specificity) of R. solani. We also evaluated the virulence of the isolates on Arabidopsis plants pretreated with salicylic acid, jasmonic acid, and ethylene. No obvious effects were detected on the symptom formation by the virulence isolates, but ethylene and salicylic acid slightly enhanced the susceptibility to the weak and nonvirulent isolates. R. solani seems to be able to overcome the induced defense by these phytohormones in the infection to Arabidopsis.

Role of Trehalose Synthesis in Ralstonia syzygii subsp. indonesiensis PW1001 in Inducing Hypersensitive Response on Eggplant (Solanum melongena cv. Senryo-nigou).

Ralstonia syzygii subsp. indonesiensis (Rsi, former name: Ralstonia solanacearum phylotype IV) PW1001, a causal agent of potato wilt disease, induces hypersensitive response (HR) on its non-host eggplant (Solanum melongena cv. Senryo-nigou). The disaccharide trehalose is involved in abiotic and biotic stress tolerance in many organisms. We found that trehalose is required for eliciting HR on eggplant by plant pathogen Rsi PW1001. In R. solanacearum, it is known that the OtsA/OtsB pathway is the dominant trehalose synthesis pathway, and otsA and otsB encode trehalose-6-phosphate (T6P) synthase and T6P phosphatase, respectively. We generated otsA and otsB mutant strains and found that these mutant strains reduced the bacterial trehalose concentration and HR induction on eggplant leaves compared to wild-type. Trehalose functions intracellularly in Rsi PW1001 because addition of exogenous trehalose did not affect the HR level and ion leakage. Requirement of trehalose in HR induction is not common in R. solanacearum species complex because mutation of otsA in Ralstonia pseudosolanacearum (former name: Ralstonia solanacearum phylotype I) RS1002 did not affect HR on the leaves of its non-host tobacco and wild eggplant Solanum torvum. Further, we also found that each otsA and otsB mutant had reduced ability to grow in a medium containing NaCl and sucrose, indicating that trehalose also has an important role in osmotic stress tolerance.

Oxicam-type non-steroidal anti-inflammatory drugs inhibit NPR1-mediated salicylic acid pathway.

Nonsteroidal anti-inflammatory drugs (NSAIDs), including salicylic acid (SA), target mammalian cyclooxygenases. In plants, SA is a defense hormone that regulates NON-EXPRESSOR OF PATHOGENESIS RELATED GENES 1 (NPR1), the master transcriptional regulator of immunity-related genes. We identify that the oxicam-type NSAIDs tenoxicam (TNX), meloxicam, and piroxicam, but not other types of NSAIDs, exhibit an inhibitory effect on immunity to bacteria and SA-dependent plant immune response. TNX treatment decreases NPR1 levels, independently from the proposed SA receptors NPR3 and NPR4. Instead, TNX induces oxidation of cytosolic redox status, which is also affected by SA and regulates NPR1 homeostasis. A cysteine labeling assay reveals that cysteine residues in NPR1 can be oxidized in vitro, leading to disulfide-bridged oligomerization of NPR1, but not in vivo regardless of SA or TNX treatment. Therefore, this study indicates that oxicam inhibits NPR1-mediated SA signaling without affecting the redox status of NPR1.

Responses of Polyamine-Metabolic Genes to Polyamines and Plant Stress Hormones in Arabidopsis Seedlings.

In plants, many of the enzymes in polyamine metabolism are encoded by multiple genes, whose expressions are differentially regulated under different physiological conditions. For comprehensive understanding of their regulation during the seedling growth stage, we examined the expression of polyamine metabolic genes in response to polyamines and stress-related plant hormones in Arabidopsis thaliana. While confirming previous findings such as induction of many of the genes by abscisic acid, induction of arginase genes and a copper amine oxidase gene, CuAOα3, by methyl jasmonate, that of an arginine decarboxylase gene, ADC2, and a spermine synthase gene, SPMS, by salicylic acid, and negative feedback regulation of thermospermine biosynthetic genes by thermospermine, our results showed that expressions of most of the genes are not responsive to exogenous polyamines. We thus examined expression of OsPAO6, which encodes an apoplastic polyamine oxidase and is strongly induced by polyamines in rice, by using the promoter-GUS fusion in transgenic Arabidopsis seedlings. The GUS activity was increased by treatment with methyl jasmonate but neither by polyamines nor by other plant hormones, suggesting a difference in the response to polyamines between Arabidopsis and rice. Our results provide a framework to study regulatory modules directing expression of each polyamine metabolic gene.

Origin of Pathogens of Grapevine Crown Gall Disease in Hokkaido in Japan as Characterized by Molecular Epidemiology of Allorhizobium vitis Strains.

Crown gall is a globally distributed and economically important disease of grapevine and other important crop plants. The causal agent of grapevine crown gall is tumorigenic Allorhizobium vitis (Ti) strains that harbor a tumor-inducing plasmid (pTi). The epidemic of grapevine crown gall has not been widely elucidated. In this study, we investigated the genetic diversity of 89 strains of Ti and nonpathogenic A. vitis to clarify their molecular epidemiology. Multi-locus sequence analysis (MLSA) of the partial nucleotide sequences of pyrG, recA, and rpoD was performed for molecular typing of A. vitis strains isolated from grapevines with crown gall symptoms grown in 30 different vineyards, five different countries, mainly in Japan, and seven genomic groups A to F were obtained. The results of MLSA and logistic regression indicated that the population of genetic group A was significantly related to a range of prefectures and that the epidemic of group A strains originated mainly in Hokkaido in Japan through soil infection. Moreover, group E strains could have been transported by infected nursery stocks. In conclusion, this study indicates that both soil infection and transporting of infected nursery stocks are working as infection source in Hokkaido.

Identification of chemoreceptor proteins for amino acids involved in host plant infection in Pseudomonas syringae pv. tabaci 6605.

Chemotaxis is crucial for Pseudomonas syringae pv. tabaci (Pta) 6605 to evoke disease in tobacco plants. Pta6605 harbors more than fifty genes for methyl-accepting chemotaxis proteins (mcp), but almost all are functionally uncharacterized. Previously we identified a dCache_1 type MCP in Pta6605 that mediates chemotaxis to γ-aminobutyric acid, called McpG. In this study, we characterized four more dCache_1 type MCPs, three of which, PscA, PscB, and PscC2, are responsible for sensing amino acids. Using a capillary chemotaxis assay, we observed that PscA, PscB, and PscC2 mutant strains had reduced chemotaxis to most amino acids, indicating that PscA and PscB mediate chemotaxis to 14 amino acids, while PscC2 has a slightly narrower ligand recognition, mediating chemotaxis to 12 amino acids. Other cellular functions were also affected in ΔpscB and ΔpscC2: swarming motility was reduced, and biofilm formation was increased. Furthermore, ΔpscB and ΔpscC2 but not ΔpscA had reduced virulence in the host tobacco plant. On the other hand, ΔpscC1 was defective in motility and did not even respond to yeast extract and was unable to cause disease. These findings supported the idea that the chemosensory pathway correlated with virulence-related phenotypes. Amino acids are abundant in tobacco apoplast; having multiple MCPs appears to support the invasion of Pta6605 into the plant.

Complete Genome Sequence of Pseudomonas amygdali pv. tabaci Strain 6605, a Causal Agent of Tobacco Wildfire Disease.

Pseudomonas amygdali pv. tabaci strain 6605 is the bacterial pathogen causing tobacco wildfire disease that has been used as a model for elucidating virulence mechanisms. Here, we present the complete genome sequence of P. amygdali pv. tabaci 6605 as a circular chromosome from reads using a PacBio sequencer.

Vfr targets promoter of genes encoding methyl-accepting chemotaxis protein in Pseudomonas syringae pv. tabaci 6605.

Virulence factor regulator (Vfr) is an indispensable transcription factor in the expression of virulence in the phytopathogenic bacteria Pseudomonas syringae. However, the function of Vfr is not known so far. The deletion of vfr resulted in the loss of surface swarming motility and reduced the virulence in P. syringae pv. tabaci (Pta) 6605. In order to identify the target genes of Vfr, we screened the sequences that bind to Vfr by chromatin immune precipitation (ChIP) and sequencing methods using the closely related bacterium P. syringae pv. syringae (Pss) B728a. For this purpose we first generated a strain that possesses the recombinant gene vfr::FLAG in Pss B728a, and performed ChIP using an anti-FLAG antibody. Immunoprecipitated DNA was purified and sequenced with Illumina HiSeq. The Vfr::FLAG-specific peaks were further subjected to an electrophoresis mobility-shift assay, and the promoter regions of locus tag for Psyr_0578 , Psyr_1776, and Psyr_2237 were identified as putative target genes of Vfr. These genes encode plant pathogen-specific methyl-accepting chemotaxis proteins (Mcp). These mcp genes seem to be involved in the Vfr-regulated expression of virulence.

Identification of novel compounds that inhibit SnRK2 kinase activity by high-throughput screening.

Abscisic acid (ABA) is a major phytohormone that regulates abiotic stress responses and development. SNF1-rerated protein kinase 2 (SnRK2) is a key regulator of ABA signaling. To isolate compounds which directly affect SnRK2 activity, we optimized a fluorescence-based system for high-throughput screening (HTS) of SnRK2 kinase regulators. Using this system, we screened a chemical library consisting of 16,000 compounds and identified ten compounds (INH1-10) as potential SnRK2 inhibitors. Further characterization of these compounds by in vitro phosphorylation assays confirmed that three of the ten compounds were SnRK2-specific kinase inhibitors. In contrast, seven of ten compounds inhibited ABA-responsive gene expression in Arabidopsis cells. From these results, INH1 was identified as a SnRK2-specific inhibitor in vitro and in vivo. We propose that INH1 could be a lead compound of chemical tools for studying ABA responses in various plant species.

Cluster II che genes of Pseudomonas syringae pv. tabaci 6605, orthologs of cluster I in Pseudomonas aeruginosa, are required for chemotaxis and virulence.

Pseudomonas syringae pv. tabaci 6605 (Pta6605) is a causal agent of wildfire disease in host tobacco plants and is highly motile. Pta6605 has multiple clusters of chemotaxis genes including cheA, a gene encoding a histidine kinase, cheY, a gene encoding a response regulator, mcp, a gene for a methyl-accepting chemotaxis protein, as well as flagellar and pili biogenesis genes. However, only two major chemotaxis gene clusters, cluster I and cluster II, possess cheA and cheY. Deletion mutants of cheA or cheY were constructed to evaluate their possible role in Pta6605 chemotaxis and virulence. Motility tests and a chemotaxis assay to known attractant demonstrated that cheA2 and cheY2 mutants were unable to swarm and to perform chemotaxis, whereas cheA1 and cheY1 mutants retained chemotaxis ability almost equal to that of the wild-type (WT) strain. Although WT and cheY1 mutants of Pta6605 caused severe disease symptoms on host tobacco leaves, the cheA2 and cheY2 mutants did not, and symptom development with cheA1 depended on the inoculation method. These results indicate that chemotaxis genes located in cluster II are required for optimal chemotaxis and host plant infection by Pta6605 and that cluster I may partially contribute to these phenotypes.

Requirement of γ-Aminobutyric Acid Chemotaxis for Virulence of Pseudomonas syringae pv. tabaci 6605.

γ-Aminobutyric acid (GABA) is a widely distributed non-proteinogenic amino acid that accumulates in plants under biotic and abiotic stress conditions. Recent studies suggested that GABA also functions as an intracellular signaling molecule in plants and in signals mediating interactions between plants and phytopathogenic bacteria. However, the molecular mechanisms underlying GABA responses to bacterial pathogens remain unknown. In the present study, a GABA receptor, named McpG, was conserved in the highly motile plant-pathogenic bacteria Pseudomonas syringae pv. tabaci 6605 (Pta6605). We generated a deletion mutant of McpG to further investigate its involvement in GABA chemotaxis using quantitative capillary and qualitative plate assays. The wild-type strain of Pta6605 was attracted to GABA, while the ΔmcpG mutant abolished chemotaxis to 10| |mM GABA. However, ΔmcpG retained chemotaxis to proteinogenic amino acids and succinic semialdehyde, a structural analog of GABA. Furthermore, ΔmcpG was unable to effectively induce disease on host tobacco plants in three plant inoculation assays: flood, dip, and infiltration inoculations. These results revealed that the GABA sensing of Pta6605 is important for the interaction of Pta6605 with its host tobacco plant.

Complete Genome Sequence Data of Nonpathogenic and Nonantagonistic Strain of Rhizobium vitis VAR06-30 Isolated From Grapevine Rhizosphere.

Rhizobium (Agrobacterium) is one genus in the family Rhizobiaceae. Most of the species are epi- or endophytic bacteria which include tumorigenic or rhizogenic pathogens, root nodule bacteria, and commensal endosymbionts. Rhizobium vitis strain VAR06-30 is a commensal bacterium without pathogenicity which was isolated from a rootstock of grapevine in Japan. It also does not have antagonistic activity to the pathogenic strain of R. vitis. Here, we show the complete genome sequence data with annotation of R. vitis VAR06-30 which was analyzed by sequence reads obtained from both PacBio and Illumina platforms. This genome sequence would contribute to the understanding of evolutionary lineage and characteristics of Rhizobium commensal bacteria.[Formula: see text] Copyright © 2020 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.