Abstract
Pathological hypertrophy (cell enlargement) plays an important role in the development of citrus canker, but its regulators are largely unknown. Although WRKY22 is known to be involved in pathogen-triggered immunity and positively regulates resistance to bacterial pathogens in Arabidopsis, rice and pepper, the CRISPR/Cas9-mediated partial knockout of CsWRKY22 improves resistance to Xanthomonas citri subsp. citri (Xcc) in Wanjincheng orange (Citrus sinensis Osbeck). Here, we demonstrate that CsWRKY22 is a nucleus-localized transcriptional activator. CsWRKY22-overexpressing plants exhibited dwarf phenotypes that had wrinkled and thickened leaves and were more sensitive to Xcc, whereas CsWRKY22-silenced plants showed no visible phenotype changes and were more resistant to Xcc. Microscopic observations revealed that the overexpression of CsWRKY22 increased cell size in the spongy mesophyll. Transcriptome analysis showed that cell growth-related pathways, such as the auxin and brassinosteroid hormonal signaling and cell wall organization and biogenesis pathways, were significantly upregulated upon CsWRKY22 overexpression. Interestingly, CsWRKY22 activated the expression of CsLOB1, which is a key gene regulating susceptibility to citrus canker. We further confirmed that CsWRKY22 bound directly to the W-boxes just upstream of the transcription start site of CsLOB1 in vivo and in vitro. We conclude that CsWRKY22 enhances susceptibility to citrus canker by promoting host hypertrophy and CsLOB1 expression. Thus, our study provides new insights into the mechanism regulating pathological hypertrophy and the function of WRKY22 in citrus.
Highlights
Citrus canker, induced by Xanthomonas citri subsp. citri (Xcc), is one of the most serious diseases facing the global citrus industry[1,2]
All yeast cells showed normal growth on the synthetic dropout medium without tryptophan (SDO), whereas only the cells transformed with GAL4BDCsWRKY22 vectors survived and turned blue when they were cultured on a selective medium supplemented with X-α-gal and aureobasidin A (SDO/X/A) (Fig. 1b)
These results suggest that the GAL4 DNA binding domain (GAL4BD)-CsWRKY22 fusion protein was able to activate the expression of the reporter genes MEL1 and AUR1, indicating that CsWRKY22 has transcriptional activation potential
Summary
Citrus canker, induced by Xanthomonas citri subsp. citri (Xcc), is one of the most serious diseases facing the global citrus industry[1,2]. The key pathogenic factors and the molecular mechanisms of the host response to Xcc are not well understood[3,4,5], which severely limits the progress of citrus disease resistance breeding. The diagnostic symptoms for citrus canker disease are hypertrophy (cell enlargement), hyperplasia (cell overdivision) and necrosis (cell death)[3]. Pathological hypertrophy and hyperplasia of host cells are the primary conditions for pustule formation and consequent canker symptoms and the spread of pathogens on the plant surface[3]. The inhibition or disruption of pathological hypertrophy and hyperplasia can efficiently repress pustule formation and pathogen spread and even confer resistance to citrus canker[6,7]; these are potential strategies for the efficient management of citrus canker. Understanding the molecular mechanisms involved in pathological hypertrophy and hyperplasia in citrus could stimulate renewed efforts to develop more effective and economical citrus canker control methods
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