Non-adapted bacterial infection suppresses plant reproduction

Abstract

Environmental stressors, including pathogens, substantially affect the growth of host plants. However, how non-adapted bacteria influence nonhost plants has not been reported. Here, we reveal that infection of Arabidopsis flowers by Xanthomonas oryzae pv. oryzae PXO99A, a bacterial pathogen causing rice blight disease, suppresses ovule initiation and reduces seed number without causing visible disease symptoms. TleB, secreted by the type VI secretion system (T6SS), interacts with plant E3 ligase PUB14 and disrupts the function of the PUB14-BZR1 module, leading to decreased ovule initiation and seed yield. On the other site, PUB14 concurrently promotes TleB's degradation. Our findings indicate that bacterial infections in nonhost plants directly repress offspring production. The regulatory mechanism by effectors PUB14-BZR1 is widely present, suggesting that plants may balance reproduction and defense and produce fewer offspring to conserve resources, thus enabling them to remain in a standby mode prepared for enhanced resistance.

Fig. 1.. Non-adapted bacterial infection inhibits ovule initiation through T6SS.

(A and B) Phenotypic analysis of placenta length and statistical analysis of ovule number per placenta after PXO99A infection. Scale bars, 100 μm. Bars represent means ± SD of three biological replicates (n = 50). (C to E) Phenotypic and statistical analysis of ovule number per pistil and silique length after PXO99A infection. Scale bars, 2 mm. Bars represent means ± SD of three biological replicates. Ovule number per pistil (n = 70) and silique length (n = 80). (F) Scanning electron microscopy (SEM) observation of bacteria on the placenta and ovule primordia. Scale bars, 5 μm. (G) Bacteria with fluorescent marker localize on ovule boundaries. Scale bars, 5 μm. (H) Phenotypic and statistical analysis of ovule number per pistil after infection of PXO99A, PXO99A (ΔT3SS), PXO99A (ΔT6SS), and PXO99A (ΔT3,6SS). Scale bars, 2 mm. Bars represent means ± SD of three biological replicates (n = 80). (I) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment and Gene Ontology (GO) term analysis of differentially expressed genes (DEGs) (PXO99A versus MgCl₂) in transcriptome analysis. Asterisks indicate significant difference (Student’s two-tailed t test, **P < 0.01). Lowercase letters indicate significant differences among different stages [one-way analysis of variance (ANOVA), P < 0.05].

Fig. 2.. TleB is ubiquitinated by PUB14 and negatively regulate ovule initiation.

(A and C) Phenotypic and statistical analysis of ovule number per pistil after infection of PXO99A and PXO99A (ΔTleA&TleB). Scale bars, 2 mm. Bars represent means ± SD of three biological replicates (n = 180). (B and D) Phenotypic and statistical analysis of ovule number per pistil of wild type (WT) and 35S:TleB-GFP. Circled are the aborted seeds of failure. Scale bars, 2 mm. Bars represent means ± SD (n = 30). (E) TleB is unstable in Arabidopsis cells (cell-free degradation assay). h, hours. (F) Relative expression of PUB14 in 35S:TleB-GFP inflorescence apices. Error bars represent confidence intervals (CIs) calculated using three technical replicates for each sample within the quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay. Asterisks indicate significant differences compared with the respective WT control (Student’s t test, *P < 0.05 and **P < 0.01). n.s., not significant. (G) Fluorescence observation of pPUB14:PUB14-GFP illustrates that PUB14 expresses in placenta and ovule primordia. Scale bars, 20 μm. Yellow dashed boxes indicate the partially enlarged tissue. White dashed boxes point to the fluorescent signal. (H) Prediction structures for PUB14-TleB interaction. (I and J) PUB14 co-localizes and directly interacts with TleB in plants (BiFC and co-IP in N. benthamiana). Scale bars, 100 μm. (K) MG132 suppresses TleB degradation in Arabidopsis cells (cell-free degradation assay). (L) TleB could be ubiquitinated by PUB14.

Fig. 3.. TleB decreases BZR1 abundance to reduce ovule initiation.

(A) Bacterial infection transcriptional regulates the expression of BES/BZRs target genes. (B to D) Bacterial infection decreases BZR1 abundance in placenta and ovule primordia. Scale bars, 20 μm. Lowercase letters indicate significant differences among different stages (one-way ANOVA, P < 0.05) (n = 15). (E) PUB14 and BZR1 co-localize in placenta and ovule primordia. Scale bars, 20 μm. Yellow dashed boxes indicate the partially enlarged tissue. White dashed boxes point to the fluorescent signal. (F) Prediction structures for PUB14-BZR1 interaction. Purple region represents the area where interactions are taking place. (G) PUB14 interacts with BZR1 directly. The dashed box circles the bands. (H) BZR1 could be mono-ubiquitinated by PUB14.

Fig. 4.. TleB inhibits the interaction of PUB14 and BZR1 and suppresses ovule initiation through competitive combining with PUB14.

(A and B) Phenotypic and statistical analysis of ovule number per placenta in WT, UBQ10:PUB14-mCherry, and PUB14 CRISPR. Bars represent means ± SD (n = 25). Scale bars, 100 μm. (C) Relative expression of PUB14 in the inflorescence apices of WT, UBQ10:PUB14-mCherry, and PUB14 CRISPR lines. Error bars represent CIs calculated using three technical replicates for each sample within the qRT-PCR assay. (D) Protein level of PUB14 in the inflorescence apices of WT, PUB14 CRISPR, and UBQ10:PUB14-mCherry lines. (E) PUB14 positive regulates BZR1 abundance in inflorescence apices. (F) DEGs related to ovule initiation in the inflorescence apices of WT, UBQ10:PUB14-mCherry, and PUB14 CRISPR lines. (G) Prediction structures for PUB14-BZR1-TleB interaction. The enlarged image shows the key sites of the PUB14-TleB interactions. (H) TleB inhibits PUB14-BZR1 interaction through competitive combining with PUB14. Lowercase letters indicate significant differences among different stages (one-way ANOVA, P < 0.05).

Fig. 5.. Mutation in the binding sites of TleB with PUB 14 decreases TleB inhibition of the interaction of PUB 14 and BZR1.

(A) TleB* also inhibits the interaction of PUB14 and BZR1 through competitive combining with PUB14, but the inhibition of TleB* is lower than that of TleB (* represent mutations at key sites in Fig. 4G, * represents 516 to 525 amino acids to A, and 727 to 743 amino acids to A in TleB). (B) The degree of PXO99A (ΔTleB TleB*)’s infection decreasing BZR1 abundance is lower than that of PXO99A. (C) The degree of PXO99A (ΔTleB TleB*)’s infection decreasing ovule initiation is lower than that of PXO99A. Bars represent means ± SD (n = 30). (D) Work model of TleB-PUB14-BZR1 module–related reproduction-defense trade-off. Lowercase letters indicate significant differences among different stages (one-way ANOVA, P < 0.05).