Arabidopsis membrane protein AMAR1 interaction with type III effector XopAM triggers a hypersensitive response.

The efficient infection of plants by the bacteria Xanthomonas campestris pv. campestris (Xcc) depends on its type III effectors (T3Es). Although the functions of AvrE family T3Es have been reported in some bacteria, the member XopAM in Xcc has not been studied. As XopAM has low sequence similarity to reported AvrE-T3Es and different reports have shown that these T3Es have different targets in hosts, we investigated the functions of XopAM in the Xcc-plant interaction. Deletion of xopAM from Xcc reduced its virulence in cruciferous crops but increased virulence in Arabidopsis (Arabidopsis thaliana) Col-0, indicating that XopAM may perform opposite functions depending on the host species. We further found that XopAM is a lipase that may target the cytomembrane and that this activity might be enhanced by its membrane-targeted protein XOPAM-ACTIVATED RESISTANCE 1 (AMAR1) in Arabidopsis Col-0. The binding of XopAM to AMAR1 induced an intense hypersensitive response that restricted Xcc proliferation. Our results showed that the roles of XopAM in Xcc infection are not the same as those of other AvrE-T3Es, indicating that the functions of this type of T3E have differentiated during long-term bacterium‒host interactions.

Ectopic Expression of HbRPW8-a from Hevea brasiliensis Improves Arabidopsis thaliana Resistance to Powdery Mildew Fungi (Erysiphe cichoracearum UCSC1).

The RPW8s (Resistance to Powdery Mildew 8) are atypical broad-spectrum resistance genes that provide resistance to the powdery mildew fungi. Powdery mildew of rubber tree is one of the serious fungal diseases that affect tree growth and latex production. However, the RPW8 homologs in rubber tree and their role of resistance to powdery mildew remain unclear. In this study, four RPW8 genes, HbRPW8-a, b, c, d, were identified in rubber tree, and phylogenetic analysis showed that HbRPW8-a was clustered with AtRPW8.1 and AtRPW8.2 of Arabidopsis. The HbRPW8-a protein was localized on the plasma membrane and its expression in rubber tree was significantly induced upon powdery mildew infection. Transient expression of HbRPW8-a in tobacco leaves induced plant immune responses, including the accumulation of reactive oxygen species and the deposition of callose in plant cells, which was similar to that induced by AtRPW8.2. Consistently, overexpression of HbRPW8-a in Arabidopsis thaliana enhanced plant resistance to Erysiphe cichoracearum UCSC1 and Pseudomonas syringae pv. tomato DC30000 (PstDC3000). Moreover, such HbRPW8-a mediated resistance to powdery mildew was in a salicylic acid (SA) dependent manner. Taken together, we demonstrated a new RPW8 member in rubber tree, HbRPW8-a, which could potentially contribute the resistance to powdery mildew.

Systematic analysis of the roles of c-di-GMP signaling in Xanthomonas oryzae pv. oryzae virulence.

Cyclic di-guanosine monophosphate (c-di-GMP) is a ubiquitous second messenger, i.e. essential to bacterial adaptation to environments. Cellular c-di-GMP level is regulated by the diguanylate cyclases and the phosphodiesterases, and the signal transduction depends on its receptors. In Xanthomonas oryzae pv. oryzae strain PXO99A, 37 genes were predicted to encode GGDEF, EAL, GGDEF/EAL, HD-GYP, FleQ, MshE, PilZ, CuxR, Clp, and YajQ proteins that may be involved in c-di-GMP turnover or function as c-di-GMP receptors. Although the functions of some of these genes have been studied, but the rest have not been extensively studied. Here, we deleted these 37 genes from PXO99A and analyzed the virulence, motility, biofilm, and EPS production of these mutants. Our results show that most of these genes are required for PXO99A virulence, motility, biofilm formation, or exopolysaccharide production. Although some of them have been reported in previous studies, we found four novel genes (gedpX8, gdpX11, pliZX4, and yajQ) are implicated in X. oryzae pv. oryzae virulence. Our data demonstrate that c-di-GMP signaling is vital for X. oryzae pv. oryzae virulence and some virulence-related factors production, but there is no positive correlation between them in most cases. Taken together, our systematic research provides a new light to understand the c-di-GMP signaling network in X. oryzae pv. oryzae.

Biosynthesis of Amino Acids in Xanthomonas oryzae pv. oryzae Is Essential to Its Pathogenicity.

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which causes a large reduction in rice production. The successful interaction of pathogens and plants requires a particular nutrient environment that allows pathogen growth and the initiation of both pathogen and host responses. Amino acid synthesis is essential for bacterial growth when bacteria encounter amino acid-deficient environments, but the effects of amino acid synthesis on Xoo pathogenicity are unclear. Here, we systemically deleted the essential genes (leuB, leuC, leuD, ilvC, thrC, hisD, trpC, argH, metB, and aspC) involved in the synthesis of different amino acids and analyzed the effects of these mutations on Xoo virulence. Our results showed that leucine, isoleucine, valine, histidine, threonine, arginine, tryptophan, and cysteine syntheses are essential to Xoo infection. We further studied the role of leucine in the interaction between pathogens and hosts and found that leucine could stimulate some virulence-related responses and regulate Xoo pathogenicity. Our findings highlight that amino acids not only act as nutrients for bacterial growth but also play essential roles in the Xoo and rice interaction.

N6-Methyladenine DNA modification in Xanthomonas oryzae pv. oryzicola genome.

DNA N6-methyladenine (6mA) modifications expand the information capacity of DNA and have long been known to exist in bacterial genomes. Xanthomonas oryzae pv. Oryzicola (Xoc) is the causative agent of bacterial leaf streak, an emerging and destructive disease in rice worldwide. However, the genome-wide distribution patterns and potential functions of 6mA in Xoc are largely unknown. In this study, we analyzed the levels and global distribution patterns of 6mA modification in genomic DNA of seven Xoc strains (BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8 and RS105). The 6mA modification was found to be widely distributed across the seven Xoc genomes, accounting for percent of 3.80, 3.10, 3.70, 4.20, 3.40, 2.10, and 3.10 of the total adenines in BLS256, BLS279, CFBP2286, CFBP7331, CFBP7341, L8, and RS105, respectively. Notably, more than 82% of 6mA sites were located within gene bodies in all seven strains. Two specific motifs for 6 mA modification, ARGT and AVCG, were prevalent in all seven strains. Comparison of putative DNA methylation motifs from the seven strains reveals that Xoc have a specific DNA methylation system. Furthermore, the 6 mA modification of rpfC dramatically decreased during Xoc infection indicates the important role for Xoc adaption to environment.

RaxM regulates the AvrXa21 (RaxX)-mediated immune response.

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease, which causes a reduction in rice production. The interaction between Xoo and rice is a model for the study of the gene-for-gene hypothesis, in which a resistance (R) gene encoding a product interacts with an effector molecule encoded by a corresponding bacterial avirulence (avr) gene. Rice XA21 functions as a plant innate immune receptor (R protein) and recognizes the avirulence protein (RaxX) of Xoo to induce the immune response and cope with pathogen attack. The sulphuration of RaxX by the tyrosine sulphotransferase RaxST is essential to its activity. The expression of raxST is regulated by the RaxH/RaxR and phoP/phoQ two-component systems. However, the regulation of raxX expression remains unclear. Here, we showed that a gene (raxM) encodes a small protein, which functions as a regulator of raxX expression. raxX and raxM are located upstream of raxST. Transcriptional analysis indicates that raxX and raxM are separately transcribed and the promoter of raxX is located at the raxM coding region. The RaxM protein regulates its own and raxX expression, and is required for the XA21-mediated immunity response. Therefore, we identified a regulator of raxX expression and of the Xoo-rice interaction. Our findings suggest that RaxX is not only regulated at the post-translational level, but also at the transcriptional level.

Genome-Wide Analysis of ß-Galactosidases in Xanthomonas campestris pv. campestris 8004.

Bacterial ß-galactosidase is involved in lactose metabolism and acts as a prevalent reporter enzyme used in studying the activities of prokaryotic and eukaryotic promoters. Xanthomonas campestris pv. campestris (Xcc) is the pathogen of black rot disease in crucifers. ß-Galactosidase activity can be detected in Xcc culture, which makes Escherichia coli LacZ unable to be used as a reporter enzyme in Xcc. To systemically understand the ß-galactosidase in Xcc and construct a ß-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative ß-galactosidases in Xcc 8004. As glycosyl hydrolase (GH) family 2 (GH2) and 35 (GH35) family enzymes were reported to have beta-galactosidase activities, we studied all of them encoded by Xcc 8004. When expressed in E. coli, only two of the enzymes, XC1214 and XC2985, were found to have ß-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no ß-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in ß-galactosidase activity. Therefore, XC1214 is the main ß-galactosidase in Xcc 8004. Notably, we have constructed a ß-galactosidase-free strain that can be employed in gene traps using LacZ as a reporter in Xcc. The results reported herein should facilitate the development of high-capacity screening assays that utilize the LacZ reporter system in Xcc.