Overexpression of miR398b impairs Verticillium wilt resistance in cotton
Expression of miR398b is differentially regulated in cotton in response to V. dahliae infection (He et al., 2014; Yin et al., 2012). Therefore, miR398b may be an important regulator in the resistance of cotton to multiple stresses. We first validated the expression patterns of miR398b by reverse transcription quantitative PCR (RT-qPCR) in various cotton tissues. The results revealed that miR398b was expressed in all tissues tested, with highest expression level in the root and anther tissues (Figure 1A). To further verify the expression pattern of miR398b, a 1,233-bp upstream fragment of miR398b was introduced into a construct upstream of the β-glucuronidase (GUS) reporter gene (Jefferson et al., 1987) and transformed into Arabidopsis for GUS expression analyses. The GUS staining pattern revealed that expression of miR398b was suppressed in the roots and cotyledons when treated with flg22, nlp20, and V. dahliae in Arabidopsis (Figure 1B). Analyses of these results indicated that the expression of miR398b was down-regulated upon V. dahlaie infection in these tissues.
To further understand the function of miR398b in cotton upon V. dahliae infection, an miR398b- overexpression vector was prepared and transformed into cotton. Two miR398b-overexpression lines with single copy insertion (O8-17, O8-18) and two null lines (ON and TN) were obtained (Figure S1A). The results of this expression analysis showed that miR398b was highly expressed in miR398b-overexpressing plants (Figure S1C). To test whether the expression level of miR398b affected the resistance of cotton to V. dahliae , the transgenic lines were inoculated with V. dahliae isolate V592. Compared to the two null lines, the miR398b-overexpressing plants were more susceptible toV. dahliae with more obvious wilt symptoms and a higher disease index than the null plants (Figure 2A, B, C). These observations were confirmed by quantification of the DNA levels of V. dahliae in the tissues, where the fungal biomass was seven-fold higher in miR398b-overexpressing plants than that in the null plants (Figure 2D). In summary, the results indicate that miR398b suppresses defense in cotton in response to V. dahliae .