PhoB-regulated phosphate assimilation of Ralstonia solanacearum is cross-activated by VsrB in Pi-abundant rich medium.

Ralstonia solanacearum is a devastating phytopathogen infecting a broad range of economically important crops. Phosphate (Pi) homeostasis and assimilation play a critical role in the environmental adaptation and pathogenicity of many bacteria. However, the Pi assimilation regulatory mechanism of R. solanacearum remains unknown. This study revealed that R. solanacearum pstSCAB-phoU-phoBR operon expression is sensitive to extracellular Pi concentration, with higher expression under Pi-limiting conditions. The PhoB-PhoR fine-tunes the Pi-responsive expression of the Pho regulon genes, demonstrating its pivotal role in Pi assimilation. By contrast, neither PhoB, PhoR, PhoU, nor PstS was found to be essential for virulence on tomato plants. Surprisingly, the PhoB regulon is activated in a Pi-abundant rich medium. Results showed that histidine kinase VsrB, which is known for the exopolysaccharide production regulation, partially mediates PhoB activation in the Pi-abundant rich medium. The 271 histidine of VsrB is vital for this activation. This cross-activation mechanism between the VsrB and PhoB-PhoR systems suggests the carbohydrate-Pi metabolism coordination in R. solanacearum. Overall, this research provides new insights into the complex regulatory interplay between Pi metabolism and growth in R. solanacearum.

Quasi-targeted metabolomics revealed isoliquiritigenin and lauric acid associated with resistance to tobacco black shank.

Tobacco black shank (TBS), caused by Phytophthora nicotianae, is a severe disease. Plant root exudates play a crucial role in mediating plant-pathogen interactions in the rhizosphere. However, the specific interaction between key secondary metabolites present in root exudates and the mechanisms of disease resistance remains poorly understood. This study conducted a comprehensive comparison via quasi-targeted metabolomic analysis on the root exudate metabolites from the tobacco cultivar Yunyan87 and K326, both before and after inoculation with P. nicotianae. The results showed that the root exudate metabolites changed after P. nicotianae inoculation, and the root exudate metabolites of different tobacco cultivar was significantly different. Furthermore, homovanillic acid, lauric acid, and isoliquiritigenin were identified as potential key compounds for TBS resistance based on their impact on the mycelium growth of the pathogens. The pot experiment showed that isoliquiritigenin reduced the incidence by 55.2%, while lauric acid reduced it by 45.8%. This suggests that isoliquiritigenin and lauric acid have potential applications in the management of TBS. In summary, this study revealed the possible resistance mechanisms of differential metabolites in resistance of commercial tobacco cultivar, and for the first time discovered the inhibitory effects of isoliquiritigenin and homovanillic acid on P. nictianae, and attempt to use plants secondary metabolites of for plant protection.

A transcriptional Regulator Gar Regulates the Growth and Virulence of Xanthomonas oryzae pv. oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of bacterial blight, one of the most devastating diseases of rice. Pathogenic bacteria possess numerous transcriptional regulators to participate in the regulation of cellular processes. Here, we identified a transcriptional regulator Gar (PXO_RS11965) that is involved in regulating the growth and virulence of Xoo. Notably, the knockout of gar in Xoo enhanced bacterial virulence to the host rice. RNA-sequencing analysis and quantitative ß-glucuronidase (GUS) assay indicated that Gar positively regulates the expression of a σ54 factor rpoN2. Further experiments confirmed that overexpression of rpoN2 restored the phenotypic changes caused by gar deletion. Our research revealed that Gar influences bacterial growth and virulence by positively regulating the expression of rpoN2.

Population genomics and pathotypic evaluation of the bacterial leaf blight pathogen of rice reveals rapid evolutionary dynamics of a plant pathogen.

The Xanthomonas oryzae pv. oryzae (Xoo) is a bacterial pathogen causing bacterial blight disease in rice, resulting in significant yield reductions of up to 50% in rice production. Despite its serious threat to food production globally, knowledge of its population structure and virulence evolution is relatively limited. In this study, we employed whole-genome sequencing to explore the diversity and evolution of Xoo in the main rice-growing areas of China over the past 30 years. Using phylogenomic analysis, we revealed six lineages. CX-1 and CX-2 primarily contained Xoo isolates from South China, while CX-3 represented Xoo isolates from North China. Xoo isolates belonging to CX-5 and CX-6 were the most prevalent across all studied areas, persisting as dominant lineages for several decades. Recent sporadic disease outbreaks were primarily caused by Xoo isolates derived from the two major lineages, CX-5 and CX-6, although Xoo isolates from other lineages also contributed to these outbreaks. The lineage and sub-lineage distributions of Xoo isolates were strongly correlated with their geographical origin, which was found to be mainly determined by the planting of the two major rice subspecies, indica and japonica. Moreover, large-scale virulence testing was conducted to evaluate the diversity of pathogenicity for Xoo. We found rapid virulence evolution against rice, and its determinant factors included the genetic background of Xoo, rice resistance genes, and planting environment of rice. This study provides an excellent model for understanding the evolution and dynamics of plant pathogens in the context of their interactions with their hosts, which are shaped by a combination of geographical conditions and farming practices. The findings of this study may have important implications for the development of effective strategies for disease management and crop protection in rice production systems.

Tn-seq identifies Ralstonia solanacearum genes required for tolerance of plant immunity induced by exogenous salicylic acid.

Ralstonia solanacearum, the causal agent of the devastating bacterial wilt disease, is of particular interest to the scientific community. The repertoire of type III effectors plays an important role in the evasion of plant immunity, but tolerance to plant immunity is also crucial for the survival and virulence of R. solanacearum. Nevertheless, a systematic study of R. solanacearum tolerance to plant immunity is lacking. In this study, we used exogenous salicylic acid (SA) to improve the immunity of tomato plants, followed by transposon insertion sequencing (Tn-seq) analysis and the identification of R. solanacearum genes associated with tolerance to plant immunity. Target gene deletion revealed that the lipopolysaccharide (LPS) production genes RS_RS02830, RS_RS03460, and RS_RS03465 are essential for R. solanacearum tolerance to plant immunity, and their expression is induced by plant immunity, thereby expanding our knowledge of the pathogenic function of R. solanacearum LPS. SA treatment increased the relative abundance of transposon insertion mutants of four genes, including two genes with unknown function, RS_RS11975 and RS_RS07760. Further verification revealed that deletion of RS_RS11975 or RS_RS07760 resulted in reduced in vivo competitive indexes but increased tolerance to plant immunity induced by SA treatment, suggesting that these two genes contribute to the trade-off between tolerance to plant immunity and fitness cost. In conclusion, this work identified and validated R. solanacearum genes required for tolerance to plant immunity and provided essential information for a more complete view of the interaction between R. solanacearum and the host plant.

Transcriptional regulator Sar regulates the multiple secretion systems in Xanthomonas oryzae.

Xanthomonas oryzae pv. oryzae (Xoo) is a notorious plant pathogen that causes leaf blight of rice cultivars. The pathogenic bacteria possess numerous transcriptional regulators to regulate various biological processes, such as pathogenicity in the host plant. Our previous study identified a new master regulator PXO_RS20790 that is involved in pathogenicity for Xoo against the host rice. However, the molecular functions of PXO_RS20790 are still unclear. Here, we demonstrate that transcriptional regulator Sar (PXO_RS20790) regulates multiple secretion systems. The RNA-sequencing analysis, bacterial one-hybrid assay, and electrophoretic mobility shift assay revealed that Sar enables binding of the promoters of the T1SS-related genes, the avirulence gene, raxX, and positively regulates these genes' expression. Meanwhile, we found that Sar positively regulated the T6SS-1 clusters but did not regulate the T6SS-2 clusters. Furthermore, we revealed that only T6SS-2 is involved in interbacterial competition. We also indicated that Sar could bind the promoters of the T3SS regulators, hrpG and hrpX, to activate these two genes' transcription. Our findings revealed that Sar is a crucial regulator of multiple secretion systems and virulence.

Genome-Wide Identification of Ralstonia solanacearum Genes Required for Survival in Tomato Plants.

Ralstonia solanacearum is an extremely destructive phytopathogenic bacterium for which there is no effective control method. Though many pathogenic factors have been identified, the survival strategies of R. solanacearum in host plants remain unclear. Transposon insertion sequencing (Tn-seq) is a high-throughput genetic screening technology. This study conducted a Tn-seq analysis using the in planta environment as selective pressure to identify R. solanacearum genes required for survival in tomato plants. One hundred thirty genes were identified as putative genes required for survival in tomato plants. Sixty-three of these genes were classified into four Clusters of Orthologous Groups categories. The absence of genes that encode the outer membrane lipoprotein LolB (RS_RS01965) or the membrane protein RS_RS04475 severely decreased the in planta fitness of R. solanacearum. RS_RS09970 and RS_RS04490 are involved in tryptophan and serine biosynthesis, respectively. Mutants that lack RS_RS09970 or RS_RS04490 did not cause any wilt symptoms in susceptible tomato plants. These results confirmed the importance of genes related to "cell wall/membrane/envelope biogenesis" and "amino acid transport and metabolism" for survival in plants. The gene encoding NADH-quinone oxidoreductase subunit B (RS_RS10340) is one of the 13 identified genes involved in "energy production and conversion," and the Clp protease gene (RS_RS08645) is one of the 11 identified genes assigned to "posttranslational modification, protein turnover, and chaperones." Both genes were confirmed to be required for survival in plants. In conclusion, this study globally identified and validated R. solanacearum genes required for survival in tomato plants and provided essential information for a more complete view of the pathogenic mechanism of R. solanacearum. IMPORTANCE Tomato plant xylem is a nutritionally limiting and dynamically changing habitat. Studies on how R. solanacearum survives in this hostile environment are important for our full understanding of the pathogenic mechanism of this bacterium. Though many omics approaches have been employed to study in planta survival strategies, the direct genome-wide identification of R. solanacearum genes required for survival in plants is still lacking. This study performed a Tn-seq analysis in R. solanacearum and revealed that genes in the categories "cell wall/membrane/envelope biogenesis," "amino acid transport and metabolism," "energy production and conversion," "posttranslational modification, protein turnover, chaperones" and others play important roles in the survival of R. solanacearum in tomato plants.

A yceI Gene Involves in the Adaptation of Ralstonia solanacearum to Methyl Gallate and Other Stresses.

Ralstonia solanacearum is a plant-pathogenic bacterium causing plant bacterial wilt, and can be strongly inhibited by methyl gallate (MG). Our previous transcriptome sequencing of MG-treated R. solanacearum showed that the yceI gene AVT05_RS03545 of Rs-T02 was up-regulated significantly under MG stress. In this study, a deletion mutant (named DM3545) and an over-expression strain (named OE3545) for yceI were constructed to confirm this hypothesis. No significant difference was observed among the growth of wild-type strain, DM3545 and OE3545 strains without MG treatment. Mutant DM3545 showed a lower growth ability than that of the wild type and OE3545 strains under MG treatment, non-optimal temperature, or 1% NaCl. The ability of DM3545 for rhizosphere colonization was lower than that of the wild-type and OE3545 strains. The DM3545 strain showed substantially reduced virulence toward tomato plants than its wild-type and OE3545 counterpart. Moreover, DM3545 was more sensitive to MG in plants than the wild-type and OE3545 strains. These results suggest that YceI is involved in the adaptability of R. solanacearum to the presence of MG and the effect of other tested abiotic stresses. This protein is also possibly engaged in the virulence potential of R. solanacearum.

Elucidation of the Pathogenicity-Associated Regulatory Network in Xanthomonas oryzae pv. oryzae.

Xanthomonas is a notorious plant pathogen causing serious diseases in hundreds of plant hosts. Xanthomonas species are equipped with an array of signal transduction systems that regulate gene expression to survive in various harsh environments and successfully infect hosts. Although certain pathogenicity-associated regulators have been functionally characterized, signal transduction systems always function as a regulatory network which remains to be elucidated in Xanthomonas This study used a systematic approach to characterize all identified pathogenicity-associated regulators in Xanthomonas oryzae pv. oryzae (Xoo), including a transcriptional regulator with unknown function, and their interactive regulatory network. RNA sequencing was used in elucidating the patterns of the 10 pathogenicity-associated regulators identified. Results revealed that each pathogenicity-associated regulator has cross talk with others and all these regulators function as a regulatory network, with VemR and PXO_RS20790 being the master pathogenicity-associated regulators and HrpX being the final executant. Moreover, regulome analysis showed that numerous genes other than genes in pathogenicity islands are finely regulated within the regulatory network. Given that most of the pathogenicity-associated regulators are conserved in Xanthomonadales, our findings suggest a global network of gene regulation in this evolutionarily conserved pathogen. In conclusion, our study provides essential basic information about the regulatory network in Xoo, suggesting that this complicated regulatory network is one of the reasons for the robustness and fitness of Xanthomonas spp.IMPORTANCE The host plant infection process of pathogenic bacteria is a coordinating cellular behavior, which requires dynamic regulation at several levels in response to variations in host plants or fluctuations in the external environment. As one of the most important genera of plant-pathogenic bacteria, Xanthomonas has been studied as a model. Although certain pathogenicity-associated regulators have been functionally characterized, interactions among them remain to be elucidated. This study systematically characterized pathogenicity-associated regulators in Xoo and revealed that cross talk exists among pathogenicity-associated regulators and function as a regulatory network in which a hierarchy exists among the regulators. Our study elucidated the landscape of the pathogenicity-associated regulatory network in Xanthomonas, promoting understanding of the infection process of pathogenic bacteria.

Cyclic-di-GMP Regulates the Quorum-Sensing System and Biocontrol Activity of Pseudomonas fluorescens 2P24 through the RsmA and RsmE Proteins.

Pseudomonas fluorescens 2P24 is a rhizosphere bacterium that protects many crop plants against soilborne diseases caused by phytopathogens. The PcoI/PcoR quorum-sensing (QS) system and polyketide antibiotic 2,4-diacetylphloroglucinol (2,4-DAPG) are particularly relevant to the strain's biocontrol potential. In this study, we investigated the effects of c-di-GMP on the biocontrol activity of strain 2P24. The expression of the Escherichia coli diguanylate cyclase (YedQ) and phosphodiesterase (YhjH) in P. fluorescens 2P24 significantly increased and decreased the cellular concentration of c-di-GMP, respectively. The production of the QS signals N-acyl homoserine lactones (AHLs) and 2,4-DAPG was negatively regulated by c-di-GMP in 2P24. The regulatory proteins RsmA and RsmE were positively regulated by c-di-GMP. Genomic analysis revealed that 2P24 has 23 predicted proteins that contain c-di-GMP-synthesizing or -degrading domains. Among these proteins, C0J56_12915, C0J56_13325, and C0J56_27925 contributed to the production of c-di-GMP and were also involved in the regulation of the QS signal and antibiotic 2,4-DAPG production in P. fluorescens Overexpression of C0J56_12915, C0J56_13325, and C0J56_27925 in 2P24 impaired its root colonization and biocontrol activities. Taken together, these results demonstrated that c-di-GMP played an important role in fine-tuning the biocontrol traits of P. fluorescensIMPORTANCE In various bacteria, the bacterial second messenger c-di-GMP influences a wide range of cellular processes. However, the function of c-di-GMP on biocontrol traits in the plant-beneficial rhizobacteria remains largely unclear. The present work shows that the QS system and polyketide antibiotic 2,4-DAPG production are regulated by c-di-GMP through RsmA and RsmE proteins in P. fluorescens 2P24. The diguanylate cyclases (DGCs) C0J56_12915, C0J56_13325, and C0J56_27925 are especially involved in regulating the biocontrol traits of 2P24. Our work also demonstrated a connection between the Gac/Rsm cascade and the c-di-GMP signaling pathway in P. fluorescens.

Different Sequevars of Ralstonia pseudosolanacearum Causing Bacterial Wilt of Bidens pilosa in China.

Bidens pilosa is an invasive weed that threatens the growth of crops and biodiversity in China. In 2017, suspected bacterial wilt of B. pilosa was discovered in Qinzhou and Beihai, Guangxi, China. A variety of weeds are considered as reservoirs harboring bacterial wilt pathogens, but most do not show obvious symptoms in the field. Identifying the classification status of the B. pilosa bacterial wilt pathogen and exploring its geographical origin might be helpful for clarifying the role of weeds in the circulation of the disease. Phylotyping, sequevar analysis, and cross inoculation of pathogens isolated from B. pilosa and nearby peanut (Arachis hypogaea), balsam gourd (Momordica charantia), and eucalyptus (Eucalyptus robusta) plants were carried out. Three isolates of B. pilosa (Bp01, Bp02, and Bp03) were identified as Ralstonia pseudosolanacearum, race 1, biovar 3, and phylotype I, and belonged to sequevars 17 and 44, and an unknown sequevar. The sequevars isolated from B. pilosa were not completely consistent with those of the nearby hosts, and the virulence of these isolates differed when cross inoculated. The Bp03 sequevar was different from peanut isolate sequevars in the same field and was not identical to any previously designated sequevars. The isolates from B. pilosa and other nearby hosts displayed low or no virulence toward their cross hosts (with wilt incidences less than 33.33%). An exception to this was the isolates from B. pilosa, which displayed high virulence toward eucalyptus (with a wilt incidence of 70.00 to 100.00%). This is the first report of different sequevars of R. pseudosolanacearum causing typical bacterial wilt symptoms in B. pilosa in the field.

The essential genome of Ralstonia solanacearum.

Ralstonia solanacearum is a scientifically/economically important plant pathogenic bacterium. The plant disease caused by R. solanacearum causes huge economic losses, and efficient control measures for the disease remain limited. To gain a better system-level understanding of R. solanacearum, we generated a near-saturated transposon insertion library of R. solanacearum GMI1000 with approximately 240,000 individual insertion mutants. Transposon sequencing (Tn-seq) allowed the mapping of 70.44%-80.96% of all potential insertion sites of the mariner C9 transposase in the genome of R. solanacearum and the identification of 465 genes essential for the growth of R. solanacearum in rich medium. Functional and comparative analyses of essential genes revealed that many basic physiological and biochemical processes such as transcription differ between R. solanacearum and other bacteria. A comparative analysis of essential genes also suggested that 34 genes might be essential only for Ralstonia group bacteria, whereas another 16 essential genes are unique to Ralstonia, providing high-priority candidate targets for developing R. solanacearum-specific drugs.

Bacterial ClpP Protease Is a Potential Target for Methyl Gallate.

Methyl gallate (MG) is an effective microbicide with great potential application in the integrated management of plant diseases and an important potential drug for clinical application. However, its target remains unknown. This study conducted a transposon sequencing (Tn-seq) under MG treatment in plant pathogenic bacterium Ralstonia solanacearum. Tn-seq identified that the mutation of caseinolytic protease proteolytic subunit gene clpP significantly increased the resistance of R. solanacearum to MG, which was validated by the in-frame gene deletion. iTRAQ (isobaric tags for relative and absolute quantitation) proteomics analysis revealed that chemotaxis and flagella associated proteins were the major substrates degraded by ClpP under the tested condition. Moreover, sulfur metabolism-associated proteins were potential substrates of ClpP and were upregulated by MG treatment in wild-type R. solanacearum but not in clpP mutant. Furthermore, molecular docking confirmed the possible interaction between MG and ClpP. Collectively, this study revealed that MG might target bacterial ClpP, inhibit the activity of ClpP, and consequently disturb bacterial proteostasis, providing a theoretical basis for the application of MG.

Bacillus thuringiensis produces the lipopeptide thumolycin to antagonize microbes and nematodes.

Bacillus thuringiensis has been widely used as a bio-insecticide. However, novel biological activities other than insect toxicity of B. thuringiensis are still underestimated. In this study, a new lipopeptide biosynthesis gene cluster in B. thuringiensis BMB171 was discovered by genome mining and verified by reverse genetics. Thumolycin, the lipopeptide synthesized by this gene cluster, was then isolated and purified. Mass spectrum analysis revealed the molecular mass of thumolycin is 696.51 Da with the predicted molecular formula of C38H64N8O4. Further bioactivities assay showed that thumolycin endowed B. thuringiensis BMB171 with broad spectrum antimicrobial and nematocidal activities.

Activation of PhoBR under phosphate-rich conditions reduces the virulence of Xanthomonas oryzae pv. oryzae.

The two-component signal transduction system PhoBR regulates the adaptation to phosphate limitation and the virulence of many animal bacterial pathogens. However, PhoBR in phytopathogens has rarely been investigated. In this study, we found that PhoBR in Xanthomonas oryzae pv. oryzae (Xoo), the pathogen of rice bacterial leaf blight, also regulates the adaptation to phosphate starvation. Unexpectedly, rice leaves infected by the phoBR-deleted mutant and wild-type PXO99A showed similar lesions, indicating that PhoBR is unnecessary for the virulence of Xoo. phoBR was found to be silenced during host infection, whereas artificially constitutive PhoBR expression attenuated virulence on host rice and growth in phosphate-rich media. RNA-sequencing (RNA-seq) was then performed to investigate the global effect caused by constitutive PhoBR activation. RNA-seq and further experiments revealed that the PhoBR regulon in Xoo comprised a wide range of genes. Nutrient transport and metabolism readjustments that resulted from PhoBR regulon activation may be responsible for growth attenuation. Our findings suggest that growth reduction regulated by PhoBR is a fitness cost of adaptation to phosphate starvation. PhoBR in Xoo is activated under phosphate-limited conditions, which could exist in epiphytic and saprophytic surviving phases, and is strictly repressed within phosphate-rich host plants to minimize fitness costs.

The global strategy employed by Xanthomonas oryzae pv. oryzae to conquer low-oxygen tension.

Xanthomonas oryzae pv. oryzae (Xoo) is a notorious rice pathogen that causes bacterial leaf blight (BLB), a destructive rice disease. Low-oxygen tension in the xylem vessels of rice stresses Xoo during infection. In this study, differentially expressed proteins under normoxic and hypoxic conditions were identified using high-performance liquid chromatography (HPLC) coupled with LC-MS/MS to investigate the global effects of low oxygen environment on Xoo PXO99A. A statistically validated list of 187 (normoxia) and 140 (hypoxia) proteins with functional assignments was generated, allowing the reconstruction of central metabolic pathways. Ten proteins involved in aromatic amino acid biosynthesis, glycolysis, butanoate metabolism, propanoate metabolism and biological adhesion were significantly modulated under low-oxygen tension. The genes encoded by these proteins were in-frame deleted, and three of them were determined to be required for full virulence in Xoo. The contributions of these three genes to important virulence-associated functions, including extracellular polysaccharide, cell motility and antioxidative ability, are presented. BIOLOGICAL SIGNIFICANCE: To study how Xanthomonas oryzae pv. oryzae (Xoo) conquers low-oxygen tension in the xylem of rice, we identified differentially expressed proteins under normoxic and hypoxia. We found 140 proteins that uniquely expressed under the hypoxia were involved in 33 metabolism pathways. We identified 3 proteins were required for full virulence in Xoo and related to the ability of extracellular polysaccharide, cell motility, and antioxidative. This study is helpful for broadening our knowledge of the metabolism processed of Xoo in the xylem of rice.

Two overlapping two-component systems in Xanthomonas oryzae pv. oryzae contribute to full fitness in rice by regulating virulence factors expression.

Two-component signal transduction systems (TCSs) are widely used by bacteria to adapt to the environment. In the present study, StoS (stress tolerance-related oxygen sensor) and SreKRS (salt response kinase, regulator, and sensor) were found to positively regulate extracellular polysaccharide (EPS) production and swarming in the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). Surprisingly, the absence of stoS or sreKRS did not attenuate virulence. To better understand the intrinsic functions of StoS and SreKRS, quantitative proteomics isobaric tags for relative and absolute quantitation (iTRAQ) was employed. Consistent with stoS and sreK mutants exhibiting a similar phenotype, the signalling circuits of StoS and SreKRS overlapped. Carbohydrate metabolism proteins and chemotaxis proteins, which could be responsible for EPS and swarming regulation, respectively, were reprogrammed in stoS and sreK mutants. Moreover, StoS and SreKRS demonstrated moderate expression of the major virulence factor, hypersensitive response and pathogenicity (Hrp) proteins through the HrpG-HrpX circuit. Most importantly, Xoo equipped with StoS and SreKRS outcompetes strains without StoS or SreKRS in co-infected rice and grows outside the host. Therefore, we propose that StoS and SreKRS adopt a novel strategy involving the moderation of Hrp protein expression and the promotion of EPS and motility to adapt to the environment.