Plant Biotechnology Journal ( IF 10.1 ) Pub Date : 2020-10-24 , DOI: 10.1111/pbi.13500 Jing Miao Liu 1 , Qiong Mei 2 , Cai Yun Xue 2 , Zi Yuan Wang 2 , Dao Pin Li 1 , Yong Xin Zhang 1 , Yuan Hu Xuan 2
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 γ subunit DEP1 (dense and erect panicle 1, Os09g26999) as a novel interactor of LPA1. The heterotrimeric G proteins, comprising α, β and γ subunits, are key players in the transmission of extracellular signals via membrane‐spanning G‐protein‐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 DNA‐binding ability of LPA1, thereby suppressing PIN1a expression, leading to an increase in planting density and resistance to sheath blight disease in rice.
中文翻译:
G蛋白γ亚基DEP1的突变增加了水稻的种植密度和对纹枯病的抗性
作物育种的重要目标之一是提高产量。在产量指标中,分蘖角度与提高光合效率和促进种植密度密切相关(Sakamoto et al ., 2006 ; Wang and Li, 2008)。具有直立分蘖、叶片和圆锥花序的水稻植株允许高密度种植系统以获得高产,但更容易发生纹枯病,从而导致产量下降。因此,作物产量与免疫途径之间的拮抗关系使得作物育种极为困难(Ning et al ., 2017)。在我们之前的研究中,我们发现松散植物结构 1 (LPA1) 的过表达通过激活 PIN1a 介导的生长素分布降低了分蘖和叶片关节角度,但增加了对纹枯病的抗性,这表明LPA1在高密度中的育种潜力种植系统(Liu et al ., 2016 ; Sun et al ., 2019)。为了进一步分析分蘖角和纹枯病调控的机制,我们进行了酵母双杂交选择,并鉴定出 G 蛋白 γ 亚基 DEP1(致密直立圆锥花序 1,Os09g26999)是 LPA1 的新型相互作用物。异源三聚体 G 蛋白,包括 α、β 和 γ 亚基,是通过跨膜 G 蛋白偶联受体将细胞外信号传递到细胞内效应器的关键参与者(Gilman,1987 年),并且圆锥花序的直立受显性等位基因控制DEP1减少了花序节间的长度 (Huang et al ., 2009)。进一步分析表明,DEP1 与全长 LPA1 及其 N 末端区域(不确定域,IDD)相互作用(图 1a)。此外,共免疫沉淀 (co-IP) 和分裂 GFP 测定证实 LPA1 与细胞核中的 DEP1 相互作用(图 1b,c)。
通过 qPCR,我们发现鞘中的DEP1表达高于叶、根和花中的表达(图 1d)。立枯丝核菌接种诱导LPA1,但不诱导DEP1(图 1e),并且 DEP1-GFP 定位于质膜和细胞核(图 1f)。为了分析粳稻品种东津中的DEP1功能,使用了将 T-DNA 插入第一个内含子的DEP1敲除突变体dep1-ko (An et al ., 2003 ) (PFG_3A-02648) 和DEP1 RNAi 系。Northern印迹结果证实DEP1在两条RNAi系(#1和#3 )中,表达被抑制了约 50%,而在dep1-ko中未检测到(图 1g)。与野生型相比,dep1-ko和DEP1 RNAi植物表现出较窄的分蘖角、相似的叶片形状和短圆锥花序(图 1h、i、j)。
先前已经表明lpa1导致更宽的分蘖角(Liu et al ., 2016 ; Wu et al ., 2013)。进一步的遗传研究表明,lpa1植物与lpa1/dep1-ko植物相似,表现出更宽的分蘖角。然而,两种基因杂合的植物(LPA1(+/-)/ DEP1(+/-))的分蘖角与野生型植物相似(图1k,l)。此外,已显示LPA1的过表达可增加对水稻纹枯病的抗性(Sun et al ., 2019)。有趣的是,dep1-ko与野生型植物相比, DEP1 RNAi植物对纹枯病的敏感性较低(图 1m,n)。经过进一步检查,我们发现lpa1和lpa1/dep1-ko植物表现出相似的症状并且更易感,而dep1-ko植物对纹枯病的敏感性明显低于野生型植物(图 1o,p)。
尽管 DEP1 与 LPA1 相互作用,但蛋白质印迹分析显示 LPA1-GFP 蛋白水平在LPA1-GFP和dep1-ko/LPA1-GFP中相似,这是一种通过LPA1-GFP和dep1-ko植物杂交的遗传组合(图 1q, r)。LPA1 通过启动子结合激活PIN1a,从而增加种植密度和对纹枯病的抗性(Sun et al ., 2019)。因此,我们通过 EMSA 和瞬时检测进一步测试了 DEP1 在 LPA1 介导的PIN1a激活中的作用。EMSA 结果表明 DEP1 抑制 LPA1 与PIN1a的结合启动子(图 1s)。通过与p35S:LPA1、p35S:DEP1或p35S:LPA1以及p35S:DEP1和在原生质体细胞中表达受pPIN1a启动子控制的 β-葡萄糖醛酸酶基因 (GUS) 的载体共转化的瞬时测定显示,共表达DEP1 的表达降低了 LPA1 刺激相对 GUS 活性的能力(图 1t),qPCR 结果也显示PIN1a在dep1-ko中的表达水平高于lpa1 、 lpa1 /dep1-ko和野生型植物(图 1u )。进一步的遗传研究表明,PIN1 RNAi植物与PIN1 RNAi / dep1-ko植物表现出更宽的分蘖角(图 1v,w)。此外,与野生型植物相比, PIN1a RNAi和PIN1 RNAi / dep1-ko植物对纹枯病的敏感性更高,而dep1-ko对纹枯病的敏感性较低(图 1x,y)。
总之,我们的分析表明,DEP1 与 LPA1 相互作用以调节PIN1a的表达,下调DEP1通过降低分蘖角度提高了种植密度,同时促进了水稻对纹枯病的抗性(图 1z)。此外,DEP1通过与 LPA1 的 N 端区域相互作用来抑制 LPA1 依赖的PIN1a转录激活,该区域是 IDD 结构域区域,一个已知的 DNA 结合结构域(Kozaki等人,2004 年)。我们的数据表明,DEP1 和 IDD 结构域之间的相互作用抑制了 LPA1 的 DNA 结合能力,从而抑制了 PIN1a表达,导致水稻种植密度和对纹枯病的抗性增加。