3.5 Involvement of on OsMYC2 in Pi starvation-enhanced JA signaling and Xoo resistance
To further discern whether OsMYC2 is involved in Pi starvation-induced Xoo resistance, we generated OsMYC2mutants using the CRISPR/Cas9 system. Two homozygous lines (myc2-3 and myc2-6 ) were obtained and confirmed by sequencing (Figure S2a). To investigate JA sensitivity, theOsMYC2 CRISPR/Cas9 mutants and NIP plants were grown with added MeJA. As expected, the transgenic lines displayed less sensitivity to MeJA than the WT plants (Figure S2b and c), indicating OsMYC2 is required for JA signaling in rice.
To investigate the role of OsMYC2 in Pi starvation signaling, we first measured the expression ofOsMYC2 -mediated JA response genes in myc2 plants upon Pi starvation. As shown in Figure 6(a), JA-responsive genes were significantly more highly expressed in Pi-starved NIP plants than in the Pi-sufficient control. Inmyc2-3 and myc2-6 plants, however, Pi deficit suppressed these Pi starvation-inducible JA-responsive genes, except forOsMYC2 itself (Figure 6a). Phytohormone measurement revealed that the production of MeJA in response to Pi starvation was abolished inmyc2 mutants (Figure 6b). JA-IIe content was lower in myc2mutants compared with NIP control, and the level of JA-IIe was not changed upon Pi starvation in both myc2 and NIP plants (Figure S1c). We also performed Xoo resistance tests on these transgenic lines grown under Pi sufficiency or starvation. 9-15 days after inoculated with Xoo , we measured bacterial proliferation of plants grown in the normal condition. The results showed that the sensitivity to Xoo in myc2-3 andmyc2-6 plants was higher than that of in NIP controls (Figure 6c). However, Pi starvation-treated myc2-3 and myc2-6 plants showed proliferation comparable to that in the control, whereas proliferation in Pi starvation-treated wild-type NIP plants was significantly reduced (Figure 6c). Blight lesion length displayed similar patterns as observed for bacterial population in myc2-3 and myc2-6 plants compared with NIP controls under both Pi-sufficient and -deficient conditions (Figure 6d and e), indicating a role for OsMYC2 in the Pi starvation-enhanced defense response. Thus, our results suggest thatOsMYC2 is necessary for Pi starvation-induced enhancement of the JA signaling and defense response.