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 .