Mutation of G‐protein γ subunit DEP1 increases planting density and resistance to sheath blight disease in rice

One of the important goals of crop breeding is yield improvement. Among the yield indices, the tiller angle is tightly associated with enhancing photosynthetic efficiency and facilitating enhanced planting density (Sakamoto et al., 2006; Wang and Li, 2008). Rice plants with erect tillers, leaves, and panicles allow a high-density planting system for high yields but are more susceptible to the occurrence of sheath blight disease causing yield reduction.

One of the important goals of crop breeding is yield improvement. Among the yield indices, the tiller angle is tightly associated with enhancing photosynthetic efficiency and facilitating enhanced planting density (Sakamoto et al., 2006;Wang and Li, 2008). Rice plants with erect tillers, leaves and panicles allow a high-density planting system for high yields but are more susceptible to the occurrence of sheath blight disease causing yield reduction. Therefore, the antagonistic relationship between crop yield and immunity pathways makes crop breeding extremely difficult (Ning et al., 2017). In our previous studies, we found that overexpression of loose plant architecture 1 (LPA1) reduced the tiller and lamina joint angle but increased resistance to sheath blight disease through activation of PIN1a-mediated auxin distribution, suggesting the breeding potential of LPA1 in high-density planting systems (Liu et al., 2016;Sun et al., 2019). To further analyse the mechanism of tiller angle and sheath blight regulation, we performed a yeast two-hybrid selection and identified G-protein c subunit DEP1 (dense and erect panicle 1, Os09g26999) as a novel interactor of LPA1. The heterotrimeric G proteins, comprising a, b and c subunits, are key players in the transmission of extracellular signals via membrane-spanning Gprotein-coupled receptors to intracellular effectors (Gilman, 1987), and panicle erectness is controlled by a dominant allele of DEP1, which reduces the length of the inflorescence internode (Huang et al., 2009). Further analysis indicated that DEP1 interacted with both full-length LPA1 and its N-terminal region (indeterminate domain, IDD) ( Figure 1a). Furthermore, coimmunoprecipitation (co-IP) and split-GFP assays confirmed that LPA1 interacted with DEP1 in the nucleus (Figure 1b,c).
Through qPCR, we found that DEP1 expression in sheaths is high compared with that in leaves, roots and flowers ( Figure 1d). Rhizoctonia solani inoculation induced LPA1, but not DEP1 (Figure 1e), and DEP1-GFP was localized at the plasma membrane and nucleus (Figure 1f). To analyse DEP1 function in the japonica rice cultivar Dongjin, a DEP1 knockout mutant dep1-ko (An et al., 2003) (PFG_3A-02648) with the T-DNA inserted into the first intron, and DEP1 RNAi lines were used. Northern blot results confirmed that DEP1 expression was suppressed by about 50% in two RNAi lines (#1 and #3) while it was not detected in dep1-ko ( Figure 1g). Compared with wild type, dep1-ko and DEP1 RNAi plants exhibited a narrow tiller angle, similar shape of leaves and a short panicle (Figure 1h,i,j).
It has previously been shown that lpa1 causes a wider tiller angle (Liu et al., 2016;Wu et al., 2013). Further genetic studies showed that lpa1 plants were similar to lpa1/dep1-ko plants exhibiting a wider tiller angle. However, the tiller angle of plants that were heterozygous for both genes (LPA1 (+/-)/DEP1 (+/-)) was similar to that of wild-type plants (Figure 1k,l). In addition, overexpression of LPA1 has been shown to increase resistance to rice sheath blight (Sun et al., 2019). Interestingly, dep1-ko and DEP1 RNAi plants were less susceptible to sheath blight compared with wild-type plants (Figure 1m,n). Upon further examination, we discovered that lpa1 and lpa1/dep1-ko plants exhibited similar symptoms and were more susceptible, while dep1-ko plants were significantly less susceptible to sheath blight than wild-type plants (Figure 1o,p).
Even though DEP1 interacts with LPA1, but Western blot analysis showed that LPA1-GFP protein levels were similar in LPA1-GFP and dep1-ko/LPA1-GFP, a genetic combination by crossing LPA1-GFP and dep1-ko plants (Figure 1q,r). LPA1 activates PIN1a via promoter binding, which increases planting density and resistance to sheath blight disease (Sun et al., 2019). Therefore, we further tested the role of DEP1 in LPA1-mediated PIN1a activation via the EMSA and transient assays. EMSA result indicated that DEP1 inhibits the binding of LPA1 to the PIN1a promoter ( Figure 1s). The transient assay by co-transformed with p35S:LPA1, p35S:DEP1 or p35S:LPA1 together with p35S:DEP1 and a vector expressing the beta-glucuronidase gene (GUS) under the control of pPIN1a promoter in protoplast cells revealed that co-expression of DEP1 reduced the ability of LPA1 to stimulate the relative GUS activity (Figure 1t), and qPCR results also showed that PIN1a expression level was higher in dep1-ko than in lpa1, lpa1/dep1-ko and wild-type plants (Figure 1u). Further genetic studies demonstrated that PIN1 RNAi plants were similar to PIN1 RNAi/dep1-ko plants exhibiting a wider tiller angle (Figure 1v,w). In addition, PIN1a RNAi and PIN1 RNAi/dep1-ko plants were more susceptible, while dep1-ko was less susceptible to sheath blight compared with wild-type plants (Figure 1x,y).
Taken together, our analyses revealed that DEP1 interacts with LPA1 to regulate PIN1a expression and that down-regulation of DEP1 enhanced planting density by decreasing the tiller angle and at the same time promoted rice resistance to sheath blight disease (Figure 1z). In addition, DEP1 inhibited LPA1-dependent activation of PIN1a transcription via interacts with the N-terminal region of LPA1, which is the IDD domain region, a known DNA-binding domain (Kozaki et al., 2004). Our data suggest that the interaction between DEP1 and the IDD domain inhibits the DNAbinding ability of LPA1, thereby suppressing PIN1a expression, leading to an increase in planting density and resistance to sheath blight disease in rice. regulating PIN1a transcription, and its mediated auxin distribution on sheath blight resistance and tiller angle. Different letters above the columns indicate a statistically significant difference between groups. The lesion areas in the leaves and sheath were calculated after 3 days following inoculation.