Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense Tropical Race 4 (Foc TR4), poses a severe threat to global banana production. Secondary metabolites are critical tools employed by pathogens to interact with their environment and modulate host–pathogen dynamics. Bikaverin, a red-colored polyketide pigment produced by several Fusarium species, has been studied for its pharmacological properties, but its ecological roles and impact on pathogenicity remain unclear. This study investigated the role of bikaverin in Foc TR4, focusing on its contribution to pathogenicity and its interaction with the rhizosphere microbiome. Pathogenicity assays under sterile and autoclaved conditions demonstrated that bikaverin does not directly contribute to pathogenicity by affecting the infection process or damaging host tissues. Instead, bikaverin indirectly enhances Foc TR4’s pathogenicity by reshaping the rhizosphere microbiome. It suppresses beneficial plant growth-promoting rhizobacteria, such as Bacillus, while promoting the dominance of fungal genera, thereby creating a microbial environment beneficial for pathogen colonization and infection. Notably, bikaverin biosynthesis was found to be tightly regulated by environmental cues, including acidic pH, nitrogen scarcity, and microbial competition. Co-culture with microbes such as Bacillus velezensis and Botrytis cinerea strongly induced bikaverin production and upregulated expression of the key bikaverin biosynthetic gene FocBik1. In addition, the identification of bikaverin-resistant Bacillus BR160, a strain with broad-spectrum antifungal activity, highlights its potential as a biocontrol agent for banana wilt management, although its stability and efficiency under field conditions require further validation. Bikaverin plays an indirect yet important role in the pathogenicity of Foc TR4 by manipulating the rhizosphere microbiome. This ecological function underscores its potential as a target for sustainable disease management strategies. Future research should focus on elucidating the molecular mechanisms underlying bikaverin-mediated microbial interactions, using integrated approaches such as transcriptomics and metabolomics. Together, these findings provide a foundation for novel approaches to combat banana wilt disease and enhance crop resistance.

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.

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.

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.

Bacterial β-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. β-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 β-galactosidase in Xcc and construct a β-galactosidase -deficient strain for promoter activity analysis using LacZ as a reporter, we here analyzed the putative β-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 β-galactosidase activity. When deleted from the Xcc 8004 genome, only the XC1214 mutant had no β-galactosidase activity, and other GH2 and GH35 gene deletions resulted in no significant reduction in β-galactosidase activity. Therefore, XC1214 is the main β-galactosidase in Xcc 8004. Notably, we have constructed a β-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.