Identification of CsLOB1 target genes that promote citrus canker disease
Final Report Abstract
Plant pathogenic bacteria inject effector proteins (effectors) into plant host cells and benefit from effector-induced changes in host cells. We study the mode of action of the transcription factor-like (TAL) effector PthA4 from Xanthomonas citri pv. citri (Xcc), the causal agent of citrus canker. Xcc without PthA4 (XccΔpthA4) show more than 10-fold reduced growth in planta. The particularly strong contribution of PthA4 to Xcc virulence was the main reason for the functional analysis of this TAL effector, even though the host plant citrus is not well suited for genetic studies. Previous work has shown that PthA4 induces increased expression of the citrus transcription factor LOB1 (CsLOB1), suggesting that Xcc exploits a CsLOB1-dependent regulon to promote its growth in the leaf apoplast. Consequently, our aim was to identify CsLOB1-regulated host genes and the CsLOB1 binding site to obtain clues on how these CsLOB1 target genes promote Xcc infection. To this end, analysis of Xcc-infected leaf tissue by RNA-seq and ChIP-seq led to the identification of ~100 direct CsLOB1 target genes, many of which encode proteins involved in cell wall degradation or expansion. In addition, a motif search revealed a 15-nucleotide CsLOB1 binding site, which was validated in vitro by electrophoretic mobility shift assays (EMSA) and in planta by promoter reporter assays. Using these promoter-reporter assays, we analysed CsLOB1-like proteins from other plant species and discovered that tomato (Solanum lycopersicum) LOB1 (SlLOB1), which has been shown to orchestrate a fruit-ripening developmental programme via tissue-specific expression, has similar or identical DNA-binding specificity as CsLOB1. Inspired by the observation that the CsLOB1-like protein, SlLOB1, is specifically expressed in ripening fruits, we investigated the native expression of CsLOB1. We found that CsLOB1, like SlLOB1, is only transcribed in ripe or maturing citrus, but not in leaves, the preferred habitat of Xcc. Since fruit ripening generally correlates with polysaccharide degradation and an increase in simple sugars, it seemed plausible that ectopic expression of CsLOB1 in leaves could condition an increased content of simple sugars in the leaf apoplast, which could serve as a carbon source for apoplast-living Xcc. Mass spectrometric analysis of apoplastic fluids obtained from Xccinfected grapefruit leaves indeed confirmed a PthA4-dependent increase in glucose, fructose and mannose. These results are consistent with a working model in which PthA4 exploits a fruit-specific developmental programme in leaves to increase the availability of Xcc-accessible carbon sources in the leaf apoplast. It is noteworthy that not only the Xcc TAL effector PthA4, but also numerous other TAL and non-TAL effectors from other microbes are known to manipulate the expression of host TFs that control developmental programmes. Our studies therefore suggest that elucidating the native, spatiotemporally controlled functions of host TFs targeted by microbial effectors is a promising route to uncovering how bacteria exploit host TF-controlled developmental programmes for their own benefit.
