Alternative splicing of rice WRKY62 and WRKY76 transcription factor genes in pathogen defense

Abstract

The WRKY family of transcription factors (TFs) functions as transcriptional activators or repressors in various signaling pathways. In this study, we discovered that OsWRKY62 and OsWRKY76, two genes of the WRKY IIa subfamily, undergo constitutive and inducible alternative splicing. The full-length OsWRKY62.1 and OsWRKY76.1 proteins formed homocomplexes and heterocomplexes, and the heterocomplex dominates in the nuclei when analyzed in Nicotiana benthamiana leaves. Transgenic overexpression of OsWRKY62.1 and OsWRKY76.1 in rice (Oryza sativa) enhanced plant susceptibility to the blast fungus Magnaporthe oryzae and the leaf blight bacterium Xanthomonas oryzae pv oryzae, whereas RNA interference and loss-offunction knockout plants exhibited elevated resistance. The dsOW62/76 and knockout lines of OsWRKY62 and OsWRKY76 also showed greatly increased expression of defense-related genes and the accumulation of phytoalexins. The ratio of full-length versus truncated transcripts changed in dsOW62/76 plants as well as in response to pathogen infection. The short alternative OsWRKY62.2 and OsWRKY76.2 isoforms could interact with each other and with full-length proteins. OsWRKY62.2 showed a reduced repressor activity in planta, and two sequence determinants required for the repressor activity were identified in the amino terminus of OsWRKY62.1. The amino termini of OsWRKY62 and OsWRKY76 splice variants also showed reduced binding to the canonical W box motif. These results not only enhance our understanding of the DNA-binding property, the repressor sequence motifs, and the negative feedback regulation of the IIa subfamily of WRKYs but also provide evidence for alternative splicing of WRKY TFs during the plant defense response.

Expression of RSOsPR10 in rice roots is antagonistically regulated by jasmonate/ethylene and salicylic acid via the activator OsERF87 and the repressor OsWRKY76, respectively

“…Based on the results of our previous work, we presumed that SA induced some OsTGA or OsWRKY proteins to repress JA/ET‐dependent RSOsPR10 expression (Takeuchi et al., ). In fact, WRKY factors are known to function not only as positive regulators but also as negative regulators by targeting a number of defense‐related genes and regulating their expression through binding to W box(es) in their promoters (Birkenbihl, Kracher, & Somssich, ; Liu, Chen, et al., ). As shown in Figures and , the 3‐kb promoter region closely mimicked the in vivo expression pattern of RSOsPR10, that is, JA/ET induction, SA suppression, and root‐specific expression.…”

“…More recent results of a study on alternative splicing variants of OsWRKY62 and OsWRKY76 transcripts indicated that short variant of OsWRKY76.2 with the LxLxLx motif at the N‐terminus exhibited repressor activity, but OsWRKY62 lacking the LxLxLx motif at the N‐terminal also had repressor activity, which was attributed to two short sequences, PTDDS and EDLEEK (Fig. ) (Liu, Chen, et al., ). Further research is required to identify the amino acid sequence motif(s) responsible for the repressor activity of these OsWRKYs and to determine which protein is the corepressor.…”

“…The gene expression of these OsWRKYs is known to be upregulated by blast fungus inoculation and benzothiadiazole (BTH) treatment, and therefore, it is probable that the regulation is downstream of the SA signaling pathway. However, it was also reported that OsWRKY76 was induced by wounding and MeJA in rice seedlings, indicating the involvement of JA signaling pathway (Liu, Chen, et al., ; Liu, Zhang, et al., ; Yokotani et al., ). These results were obtained with leaves and shoots, but studies with roots have not been performed so far.…”

“…Most WRKYs bind to a consensus cis element known as the W box ((T/C)TGAC(C/T)) and mainly act as positive transcription factors, although group IIa WRKYs exhibit negative functions. In rice, the WRKY IIa subfamily has four members (OsWRKY28, OsWRKY62, OsWRKY71, and OsWRKY76), at least three of which (OsWRKY28, OsWRKY62, and OsWRKY76) function as transcriptional repressors in blast disease resistance (Chujo et al., ; Fukushima, Mori, Sugano, & Takatsuji, ; Liu, Chen, et al., ; Liu, Zhang, et al., ; Peng, Bartley, Canlas, & Ronald, ; Yokotani et al., ). The gene expression of these OsWRKYs is known to be upregulated by blast fungus inoculation and benzothiadiazole (BTH) treatment, and therefore, it is probable that the regulation is downstream of the SA signaling pathway.…”

Molecular characters and different expression of WRKY1 gene from Gossypium barbadense L. and Gossypium hirsutum L.

“…oryzae. [10] Similarly, transgenic lines with the GhWRKY27a gene exhibited more severe disease symptoms after Rhizoctonia solani infection in Nicotiana benthamiana. [11] By contrast, mutations of AtWRKY70 reduced the plant tolerance to the bacterial pathogen Erwinia carotovora as well as to the fungal pathogens Erysiphe cichoracearum and Botrytis.…”