Predation of oomycetes by myxobacteria via a specialized CAZyme system arising from adaptive evolution.

As social micropredators, myxobacteria are studied for their abilities to prey on bacteria and fungi. However, their predation of oomycetes has received little attention. Here, we show that Archangium sp. AC19 secretes a carbohydrate-active enzyme (CAZyme) cocktail during predation on oomycetes Phytophthora. These enzymes include three specialized ß-1,3-glucanases (AcGlu13.1, -13.2 and -13.3) that act as a cooperative consortium to target ß-1,3-glucans of Phytophthora. However, the CAZymes showed no hydrolytic effects on fungal cells, even though fungi contain ß-1,3-glucans. Heterologous expression of AcGlu13.1, -13.2 or -13.3 enzymes in Myxococcus xanthus DK1622, a model myxobacterium that antagonizes but does not predate on P. sojae, conferred a cooperative and mycophagous ability that stably maintains myxobacteria populations as a mixture of engineered strains. Comparative genomic analyses suggest that these CAZymes arose from adaptive evolution among Cystobacteriaceae myxobacteria for a specific prey killing behavior, whereby the presence of Phytophthora promotes growth of myxobacterial taxa by nutrient release and consumption. Our findings demonstrate that this lethal combination of CAZymes transforms a non-predatory myxobacterium into a predator with the ability to feed on Phytophthora, and provides new insights for understanding predator-prey interactions. In summary, our work extends the repertoire of myxobacteria predatory strategies and their evolution, and suggests that these CAZymes can be engineered as a functional consortium into strains for biocontrol of Phytophothora diseases and hence crop protection.

Hfq, a RNA Chaperone, Contributes to Virulence by Regulating Plant Cell Wall-Degrading Enzyme Production, Type VI Secretion System Expression, Bacterial Competition, and Suppressing Host Defense Response in Pectobacterium carotovorum.

Hfq is a RNA chaperone and participates in a wide range of cellular processes and pathways. In this study, mutation of hfq gene from Pectobacterium carotovorum subsp. carotovorum PccS1 led to significantly reduced virulence and plant cell wall-degrading enzyme (PCWDE) activities. In addition, the mutant exhibited decreased biofilm formation and motility and greatly attenuated carbapenem production as well as secretion of hemolysin coregulated protein (Hcp) as compared with wild-type strain PccS1. Moreover, a higher level of callose deposition was induced in Nicotiana benthamiana leaves when infiltrated with the mutant. A total of 26 small (s)RNA deletion mutants were obtained among a predicted 27 sRNAs, and three mutants exhibited reduced virulence in the host plant. These results suggest that hfq plays a key role in Pectobacterium virulence by positively impacting PCWDE production, secretion of the type VI secretion system, bacterial competition, and suppression of host plant responses.

AsnB, regulated by diffusible signal factor and global regulator Clp, is involved in aspartate metabolism, resistance to oxidative stress and virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola (Xoc) causes bacterial leaf streak in rice, which is a destructive disease worldwide. Xoc virulence factors are regulated by diffusible signal factor (DSF) and the global regulator Clp. In this study, we have demonstrated that asnB (XOC_3054), encoding an asparagine synthetase, is a novel virulence-related gene regulated by both DSF and Clp in Xoc. A sequence analysis revealed that AsnB is highly conserved in Xanthomonas. An asnB mutation in Xoc dramatically impaired pathogen virulence and growth rate in host rice, but did not affect the ability to trigger the hypersensitive response in nonhost (plant) tobacco. Compared with the wild-type strain, the asnB deletion mutant was unable to grow in basic MMX (-) medium (a minimal medium without ammonium sulphate as the nitrogen source) with or without 10 tested nitrogen sources, except asparagine. The disruption of asnB impaired pathogen resistance to oxidative stress and reduced the transcriptional expression of oxyR, katA and katG, which encode three important proteins responsible for hydrogen peroxide (H(2)O(2)) sensing and detoxification in Xanthomonas in the presence of H(2)O(2), and nine important known Xoc virulence-related genes in plant cell-mimicking medium. Furthermore, the asnB mutation did not affect extracellular protease activity, extracellular polysaccharide production, motility or chemotaxis. Taken together, our results demonstrate the role of asnB in Xanthomonas for the first time.

epv, Encoding a hypothetical protein, is regulated by DSF-mediating quorum sensing as well as global regulator Clp and is required for optimal virulence in Xanthomonas oryzae pv. oryzicola.

Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak in rice, a destructive disease worldwide. In this study, six putative hypothetical secreted proteins, which were absent in X. oryzae pv. oryzae, were detected from X. oryzae pv. oryzicola strain BLS256. Disruption-based mutagenesis study revealed that one of them, Xoc_15235, named as extracellular polysaccharide and virulence-related gene (epv), was required for the optimal virulence in host rice but not for the induction of a hypersensitive reaction in nonhost tobacco. Sequence analysis revealed that epv was highly conserved in Xanthomonas spp. (except X. oryzae pv. oryzae). In-frame deletion of epv in X. oryzae pv. oryzicola dramatically impaired pathogen virulence and extracellular polysaccharide (EPS) production, one of the important known virulence-associated functions in Xanthomonas spp. Quantitative real-time reverse-transcription polymerase chain reaction showed that expression of both gumB (a gene encoding exopolysaccharide xanthan biosynthesis export protein) and a known virulence-related gene, pgk (encoding phosphoglycerate kinase), were obviously reduced in the epv-deletion mutant compared with the wild-type strain Rs105. In addition, we observed that epv was positively regulated by both diffusible signal factor and global regulator Clp in X. oryzae pv. oryzicola. Taken together, the novel roles and genetics of epv of X. oryzae pv. oryzicola in the EPS production and virulence were investigated for the first time.

[Analysis of the flgD and figE genes regulated by diffusible signal factor in Xanthomonas oryzae pv. oryzicola].

OBJECTIVE: To investigate functions of flgDxoc and flgExoc genes regulated by diffusible signal factor (DSF) in Xanthomonas oryzae pv. oryzicola(Xoc)Rs105. METHODS: TheflgDxoc and flgExoc genes were amplified by PCR. We constructed deltaflgDxoc and deltaflgExoc, the deletion mutants from Rs105 by using double crossover method, and determined cell morphology, motility, pathogenicity in host rice and hypersensitive response (HR) in nonhost tobacco. We tested the differential expression of flgDxoc and flgExoc gene by reverse transcriptional polymerase chain reaction (RT-PCR) between the wide type and deltarpfFxoc (the deletion mutant of rpfFxoc gene, which could not produce DSF). RESULTS: We cloned flgDxoc and flgExoc from genomic DNA of Rs105. PCR and Southern blot analysis demonstrated that the flgDxoc and flgExoc genes were knocked out successfully. Both mutants were non-flagellated and significantly attenuated motility on the 0.3% semi-solid medium. The pathogenicity on rice were obviously attenuated in deltaflgDxoc and deltaflgExoc compared to the wild type. All the changes in mutant could be restored through the complementation. However, there was no significant difference in bacterial growth in MMX medium and induction of HR between mutant (deltaflgDxoc or deltaflgExoc) and the wild type. In addition, the results of RT-PCR demonstrated that the transcription level of flgDxoc and flgExoc were downregulated in deltarpfFxoc. CONCLUSION: This study showed that expressions of flgDxoc and flgExoc were positively regulated by DSF, and necessary for flagellar hook assembly and flagellar structure in Xoc. Meanwhile, FlgD and FlgE contributed to pathogen's virulence, motility and chemotaxis, but no differences at growth rate in MMX medium and HR in nonhost. In addition, our results provided molecular evidences that the contribution of DSF-type quorum sensing to pathogen's virulence might be, at least partially, dependent on bacterial flagellar in Xoc.