Antagonistic transcriptome profile reveals potential mechanisms of action on Xanthomonas oryzae pv. oryzicola by the cell-free supernatants of Bacillus velezensis 504, a versatile plant probiotic bacterium

Abstract

Bacterial leaf streak (BLS) of rice is a severe disease caused by the bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) that has gradually become the fourth major disease on rice in some rice-growing regions in southern China. Previously, we isolated a Bacillus velezensis strain 504 that exhibited apparent antagonistic activity against the Xoc wild-type strain RS105, and found that B. velezensis 504 was a potential biocontrol agent for BLS. However, the underlying mechanisms of antagonism and biocontrol are not completely understood. Here we mine the genomic data of B. velezensis 504, and the comparative transcriptomic data of Xoc RS105 treated by the cell-free supernatants (CFSs) of B. velezensis 504 to define differentially expressed genes (DEGs). We show that B. velezensis 504 shares over 89% conserved genes with FZB42 and SQR9, two representative model strains of B. velezensis, but 504 is more closely related to FZB42 than SQR9, as well as B. velezensis 504 possesses the secondary metabolite gene clusters encoding the essential anti-Xoc agents difficidin and bacilysin. We conclude that approximately 77% of Xoc RS105 coding sequences are differentially expressed by the CFSs of B. velezensis 504, which significantly downregulates genes involved in signal transduction, oxidative phosphorylation, transmembrane transport, cell motility, cell division, DNA translation, and five physiological metabolisms, as well as depresses an additional set of virulence-associated genes encoding the type III secretion, type II secretion system, type VI secretion system, type IV pilus, lipopolysaccharides and exopolysaccharides. We also show that B. velezensis 504 is a potential biocontrol agent for bacterial blight of rice exhibiting relative control efficiencies over 70% on two susceptible cultivars, and can efficiently antagonize against some important plant pathogenic fungi including Colletotrichum siamense and C. australisinense that are thought to be the two dominant pathogenic species causing leaf anthracnose of rubber tree in Hainan province of China. B. velezensis 504 also harbors some characteristics of plant growth-promoting rhizobacterium such as secreting protease and siderophore, and stimulating plant growth. This study reveals the potential biocontrol mechanisms of B. velezensis against BLS, and also suggests that B. velezensis 504 is a versatile plant probiotic bacterium.

Keywords: Bacillus velezensis; Xanthomonas oryzae pv. oryzicola; antagonism mechanism; biocontrol agent; transcriptome profiling.

Figure 1

The complete genome map of B. velezensis 504 with its genomic features. (A) Circular map of B. velezensis 504 chromosome. The GC content of the strain 504 chromosome was 46.6% and contained 3,853 protein coding sequences (CDSs), 27 rRNA genes, 86 tRNA genes and 217 other non-coding RNA genes. (B) Circular map of B. velezensis 504 plasmid. The GC content of the 8,564 bp-plasmid was 39.86% and different from B. velezensis strains FZB42 and SQR9.

Figure 2

Comparative genomic analysis of B. velezensis 504 with other B. velezensis strains (B. velezensis FZB42 and SQR9). (A) Genome-to-genome alignment of B. velezensis 504, B. velezensis FZB42, and B. velezensis SQR9 using a progressive mauve software with B. velezensis 504 as the reference genome. Similar-colored boxes represent syntenic regions. Boxes below the horizontal line represent regions that are inverted. Rearrangements are depicted with colored lines. (B) Venn diagram illustrating the number of orthologous CDS genes that are shared and unique among three strains of B. velezensis 504, B. velezensis FZB42, and B. velezensis SQR9. (C) Gene clusters of secondary metabolite biosynthesis in B. velezensis 504, B. velezensis FZB42, and B. velezensis SQR9. The number represented the Jaccard index on the horizontal line. Connected by black lines are the colinear regions. T3PKS, type III PKS; NRPS, non-ribosomal peptide synthetase cluster.

Figure 3

Overview of transcriptome of RS105 under B. velezensis 504 CFSs treatment. (A) Schematic presentation of the transcriptome assay of B. velezensis 504 CFS-treated RS105. (B) Bar chart of downregulated and upregulated DEGs distribution between RS105 and 504-treated RS105 at three distinct time points. (C–E) Gene expression patterns obtained through RNA-Seq and qRT-PCR validation have been compared. The Y-axis displays the Log₂FCs of the DEGs obtained using qRT-PCR validation, whereas the X-axis represents the Log₂FCs of the DEGs obtained using RNA-Seq. The equation described the correlation between X and Y. (F) Venn diagram showing the overlapping down-regulated DEGs at three distinct time points. (G) Venn diagram showing the overlapping up-regulated DEGs at three distinct time points.

Figure 4

Defining the transcriptional response of RS105 to the CFSs of B. velezensis 504. (A) Heatmap showing the temporal pattern of the relative transcript abundance of nucleotide metabolism genes between RS105 and the CFSs of 504-treated RS105. (B) Heatmap showing the temporal pattern of the relative transcript abundance of carbohydrate metabolism genes between RS105 and the CFSs of 504-treated RS105. (C) Heatmap showing the temporal pattern of the relative transcript abundance of two-component genes between RS105 and the CFSs of 504-treated RS105. (D) Heatmap showing the temporal pattern of the relative transcript abundance of oxidative phosphorylation genes between RS105 and the CFSs of 504-treated RS105. (E) Heatmap showing the temporal pattern of the relative transcript abundance of cell motility and biofilm formation genes between RS105 and the CFSs of 504-treated RS105. (F) Heatmap showing the temporal pattern of the relative transcript abundance of translation genes between RS105 and the CFSs of 504-treated RS105. (G) Heatmap showing the temporal pattern of the relative transcript abundance of ABC transporters genes between RS105 and the CFSs of 504-treated RS105. (H) Heatmap showing the temporal pattern of the relative transcript abundance of Amino acid metabolism genes between RS105 and the CFSs of 504-treated RS105. (I) Heatmap showing the temporal pattern of the relative transcript abundance of amino sugar and nucleotide sugar metabolism genes between RS105 and the CFSs of 504-treated RS105. (J) Heatmap showing the temporal pattern of the relative transcript abundance of cell division genes between RS105 and the CFSs of 504-treated RS105.

Figure 5

qRT-PCR assays for gene expression in RS105 treated with B. velezensis 504 CFSs. (A–O) The X-axis represents the 6, 12, and 24hpt. The Y-axis represents the Log₂FoldChange (Untreated vs the CFSs of 504-treated) of relative expression level determined by RNA-Seq and qRT-PCR using rpoD and gyrB as an endogenous control. All experiments were repeated three times with consistent results.

Figure 6

Defining the transcriptional response of RS105 virulence genes to the CFSs of B. velezensis 504 treatment. (A) Heatmap showing the temporal pattern of the relative transcript abundance of T3SS genes between RS105 and the CFSs of 504-treated RS105. (B) Heatmap showing the temporal pattern of the relative transcript abundance of T4P genes between RS105 and the CFSs of 504-treated RS105. (C) Heatmap showing the temporal pattern of the relative transcript abundance of T6SS genes between RS105 and the CFSs of 504-treated RS105. (D) Heatmap showing the temporal pattern of the relative transcript abundance of other genes related with virulence between RS105 and the CFSs of 504-treated RS105.

Figure 7

Efficacy of B. velezensis 504 for control of Xoo PXO99^A in the field. The field trials were performed using the susceptible cultivars including indica rice IR24 (A) and japonica cultivar Yuanfengzao (B). The prevention (Pre) and treatment (Tre) strategies were executed as follows: Xoo PXO99^A only (Control), rice leaves sprayed with B. velezensis 504 12 h before inoculation with Xoo PXO99^A suspension (504-Pre), and 12 h after inoculation with Xoo PXO99^A suspension (504-Tre). The bacterial blight disease of rice (BB) severity was investigated after 15 days. Data points represent means ± SD (n=8 independent leaves). The significant differences at ***P< 0.001 and at ****P< 0.0001.

Figure 8

B. velezensis 504 can efficiently antagonize against some important plant pathogenic fungi and promote plant growth. (A) Determination of B. velezensis 504 antagonistic activity against F. graminearum, B. cinerea, M.oryzae, F. oxysporum in co-cultured assays. Sterile supernatants and fermented liquid of B. velezensis 504 were used as treatment, blank was used as control (CK). Three independent biological experiments were performed with similar results. (B) Determination of B. velezensis 504 antagonistic activities against C. siamense and C. australisinense in co-cultured assays. (C) Effect of B. velezensis 504 on growth-promotion of pak choi. (D) Siderophores and protease production of B. velezensis 504. The significant differences at ****P< 0.0001.