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Plant secondary metabolites perform numerous functions in the interactions between plants and pathogens. However, little is known about the precise mechanisms underlying their contribution to the direct inhibition of pathogen growth and virulence in planta. Here, we show that the secondary metabolite sulforaphane (SFN) in crucifers inhibits the growth, virulence, and ability of Xanthomonas species to adapt to oxidative stress, which is essential for the successful infection of host plants by phytopathogens. The transcription of oxidative stress detoxification-related genes (catalase [katA and katG] and alkylhydroperoxide-NADPH oxidoreductase subunit C [ahpC]) was substantially inhibited by SFN in Xanthomonas campestris pv. campestris (Xcc), and this phenomenon was most obvious in sax gene mutants sensitive to SFN. By performing microscale thermophoresis (MST) and electrophoretic mobility shift assay (EMSA), we observed that SFN directly bound to the virulence-related redox-sensing transcription factor OxyR and weakened the ability of OxyR to bind to the promoters of oxidative stress detoxification-related genes. Collectively, these results illustrate that SFN directly targets OxyR to inhibit the bacterial adaptation to oxidative stress, thereby decreasing bacterial virulence. Interestingly, this phenomenon occurs in multiple Xanthomonas species. This study provides novel insights into the molecular mechanisms by which SFN limits Xanthomonas adaptation to oxidative stress and virulence, and the findings will facilitate future studies on the use of SFN as a biopesticide to control Xanthomonas.

Bacteria often use multiple transcription factors to regulate specific biological processes. Biosynthesis of heat-stable antifungal factor (HSAF) is regulated by multiple factors in Lysobacter enzymogenes. However, the mechanism of HSAF biosynthesis regulation remains largely unknown. In this study, we screened a potential HSAF biosynthesis regulator, RecX, by a DNA pull-down assay. Deletion of recX resulted in a significant increase in the production of HSAF, and overexpression of recX significantly suppressed HSAF production. Importantly, our results showe that RecX directly binds to the promoter region of the lafB gene to inhibit its transcription and thus decreases HSAF production in L. enzymogenes. These findings reveal the novel mechanism of RecX regulation of antifungal antibiotic production in L. enzymogenes.

Anthracnose, caused by fungi of the genus Colletotrichum, is a serious disease of chili worldwide. Sterol 14α-demethylation inhibitors (DMIs) are a class of chemical fungicides that can effectively control anthracnose diseases. In this study, 22 Colletotrichum isolates collected from commercial chili fields in Zhangzhou, Fujian Province, China, were identified as Colletotrichum siamense. The sensitivities of the 22 C. siamense isolates to tebuconazole were determined based on the EC50 (50% effective inhibition concentration) value. The results showed that the EC50 values of the two isolates to tebuconazole were 0.039 ± 0.0036 and 0.042 ± 0.0012 mg/L, while the other 20 isolates showed significantly decreased sensitivities to tebuconazole, with EC50 values ranging from 0.61 ± 0.056 to 1.94 ± 0.11 mg/L. Sequence analysis of CYP51A and CYP51B revealed five genotype mutations (i. e., CYP51AV46L, D115V, P163S, R306K, E397D, CYP51AD115V, S164Y, R306K, E397D, CYP51AD115V, R306K, P339T, E397D, CYP51AD115V, R306K, E397D, S400N, and CYP51AD115V, R306K, E397DCYP51BR266H) in the resistant isolates. The tebuconazole-resistant isolates of five genotypes suffered a fitness penalty and exhibited cross-resistance to difenoconazole, prochloraz, and propiconazole. Additionally, the five genotype mutations were validated as being responsible for tebuconazole-resistance in C. siamense by construction of replacement mutants. Overexpression of CYP51A and CYP51B was not detected in the replacement mutants of the five genotypes. Overall, the present study is the first to report DMI resistance in C. siamense and provides significant information for rational use of DMIs to control chili anthracnose.

Soil bacteria often provide multiple weapons for eukaryotes or prokaryotes to use against predators. Diffusible signal factors (DSFs) represent a unique group of quorum sensing (QS) chemicals that modulate interspecies competition in bacteria that do not produce antibiotic-like molecules. However, the molecular mechanism by which DSF-mediated QS systems regulate weapons production for interspecies competition remains largely unknown in soil biocontrol bacteria. In this study, we found that the necessary QS system component protein RpfG from Lysobacter, in addition to being a cyclic dimeric GMP (c-di-GMP) phosphodiesterase (PDE), regulates the biosynthesis of an antifungal weapon (heat-stable antifungal factor, HSAF), which does not appear to depend on the enzymatic activity. Interestingly, we showed for the first time that RpfG interacts with three hybrid two-component system (HyTCS) proteins, HtsH1, HtsH2, and HtsH3, to regulate HSAF production in Lysobacter. In vitro studies showed that each of these proteins interacted with RpfG, which reduced the PDE activity of RpfG. Finally, we showed that the cytoplasmic proportions of these proteins depended on their phosphorylation activity and binding to the promoter controlling the genes implicated in HSAF synthesis. These findings reveal a new mechanism of DSF signalling in antifungal weapon production in soil bacteria.

Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.

Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria., In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes. IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.

Colletotrichum fructicola, the causal agent of pear anthracnose, causes significant annual economic losses. Mitogen-activated protein kinase (MAPK) cascades are highly conserved signal transduction pathways that play a crucial role in mediating cellular responses to environmental and host signals in plant pathogenic fungi. In this study, we identified an ortholog of the FUS3/KSS1-related MAPK gene, CfMK1, and characterized its function in C. fructicola. The Cfmk1 deletion mutants exhibited poorly developed aerial hyphae, autolysis, no conidial mass or perithecia on solid plates. However, the conidiation of the Cfmk1 mutant in PDB liquid medium was normal compared with that of the wild type (WT). Conidia of the Cfmk1 mutant exhibited a reduced germination rate on glass slides or plant surfaces. The Cfmk1 deletion mutants were unable to form appressoria and lost the capacity to penetrate plant epidermal cells. The ability of the Cfmk1 mutants to infect pear leaves and fruit was severely reduced. Moreover, RNA sequencing (RNA-seq) analysis of the WT and Cfmk1 mutant was performed, and the results revealed 1886 upregulated and 1554 downregulated differentially expressed genes (DEGs) in the mutant. The DEGs were significantly enriched in cell wall and pathogenesis terms, which was consistent with the defects of the Cfmk1 mutant in cell wall integrity and plant infection. Overall, our data demonstrate that CfMK1 plays critical roles in the regulation of aerial hyphal growth, asexual and sexual reproduction, autolysis, appressorium formation, and pathogenicity.

Among Lysobacter species, Lysobacter antibioticus has been demonstrated to be an interesting source of antimicrobial metabolites for the biocontrol of plant diseases. Although the antibacterial activity was attributed to N-oxide phenazines, the active compounds involved in the antifungal function remained unknown. In this work, an antifungal compound was isolated and identified as p-aminobenzoic acid (pABA). Antifungal activity screening revealed that pABA shows activity against a number of plant pathogens. The genes involved in the synthetic route of this compound in OH13 were identified. Further, the production of pABA was optimized by modification of the carbon source using engineered L. antibioticus OH13 strains.