Rice (Oryza sativa) is a staple food supply for over half of the global population. Various phytopathogens including viruses pose a significant threat to rice yield and quality. Southern rice black-streaked dwarf virus (SRBSDV), belonged to the genus Fijivirus, family Reoviridae, has become a major virus species leading to substantial crop losses in Asian nations (Zhang et al., 2023). Traditional breeding and commercial rice varieties face challenges in achieving viral resistance due to the absence of natural resistance. Therefore, it is crucial to utilize biotechnology methods to create and cultivate resistant germplasm for the prevention and control of viral diseases.

Oxygenic photosynthesis is the primary process that converts sunlight into chemical energy in higher plants. The light reaction of photosynthesis is driven by photosystems I and II (PSI and PSII). PSI is a membrane protein complex that enables sunlight-driven transmembrane electron transfer as a component of the photosynthetic machinery (Malavath et al., 2018; Varotto et al., 2000). As a component of PSI, PsaL is crucial for the formation of PSI trimers, a process likely reliant on the binding of calcium ions to the PsaL subunit. However, the involvement of PsaL in plant growth and immunity remains unclear.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) technology have been effectively utilized to create new cultivars from wild species via de novo domestication (Bai et al., 2023). In this study, we demonstrate the successful application of CRISPR/Cas9 in rice to create the transgenic lines with superior agronomic traits and resistance to SRBSDV. We firstly found that the expression level of OsPsaL gene was significantly down-regulated following SRBSDV infection (Figure 1a). Then, we generated two independent ospsal-ko mutants (ospsal-1 and ospsal-2) via the CRISPR/Cas9 system in the Nipponbare (NIP) background (Figure 1b,c). Subsequently, we used chlorophyll fluorescence to assess the photosynthetic traits of transgenic plants. In contrast to the wild type, the electron transport rate (ETR) and net photosynthetic efficiency (pN) notably increased, while Y(NO), an indicator of unregulated heat dissipation and fluorescence, decreased as light intensity rose in ospsal-ko (Figure 1d–f), indicating that photosynthesis has been enhanced in the mutant. We further constructed overexpressing OsPsaL-transgenic rice named OsPsaL-ox (OsPsaL-3# and OsPsaL-4#) (Figure S1a,b). OsPsaL-ox plants displayed a decreased electron transport rate and net photosynthetic efficiency but exhibited no variance in Y (NO) compared to wild type (Figure S1c–e). Statistical analysis revealed that ospsal-ko has a higher number of tillers, panicles, and grains per plant, but they did exhibit no difference in seed size and 1000 grain weight or proportion of amylose compared to wild-type plants (Figure 1g–l). The grain length and 1000 grain weight of OsPsaL-3 were significantly higher compared with wild-type plants (Figure S1f,g). However, OsPsaL-overexpressing plants exhibited significantly reduced tillers and panicles (Figure S1h,i); therefore, the overall yield of the plants was obviously reduced (Figure S1j). These findings suggest that ospsal-ko photosynthesis is enhanced, and yield is increased.

Next, we aim to investigate the role of OsPsaL in SRBSDV infection. After inoculated with SRBSDV about 30 days, the ospsal-ko showed less dwarfed than the controls (Figure 1m). Virus content detection showed that the contents of viral coat protein P10 and SRBSDV RNAs (S2, S4 and S6) were markedly reduced in the mutant ospsal-ko compared to the controls (Figure 1n,o). While OsPsaL-ox plants exhibited more severe dwarfing and higher accumulations of virus in both RNA and protein levels compared to the wild-type plants (Figure S1l–n). Together, these results suggest that OsPsaL plays a negative role in rice defence against SRBSDV. To explore the broad-spectrum disease resistance of ospsal-ko, we inoculated the transgenic plants with a different type of rice virus (Rice stripe virus, RSV), revealing that the ospsal-ko also exhibited resistance to RSV while OsPsaL-ox showed higher sensitivity to RSV (Figure 1p–r and Figure S1o–q).

We further performed transcriptome sequencing on ZH11 and OsPsaL-ox in response to SRBSDV infection. Examined the differentially expressed genes with specific expression in the comparisons OsPsaL-3#-V versus OsPsaL-3#-H but not found in ZH11-V versus ZH11-H, resulting in the identification of 2178 genes (Figure S1r). These genes were mostly suppressed in SRBSDV-infected OsPsaL-3# plants compared with ZH11. GO analysis showed that these down-regulated genes were highly enriched in photosynthesis (Figure S1s). These findings indicate that the photosynthesis of OsPsaL-ox rice is significantly impaired by SRBSDV. Moreover, a comprehensive analysis of the transcriptome showed a significant downregulation of several jasmonic acid (JA)-related genes in OsPsaL-3# compared to ZH11 (Figure S1t). JA is commonly recognized as the essential antiviral pathway (Li et al., 2021; Zhang et al., 2023). Further RT-qPCR assays showed that the expression JA-related genes (OsLOX2, OsAOC, OsAOS2 and OsJMT1) were significantly activated in ospsal-ko but repressed in OsPsaL-ox compared to the wild-type plants (Figure 1s; Figure S1u–x). JA contents assays showed that the JA concentration was significantly higher in ospsal-ko but lower in OsPsaL-ox than in wild-type plants (Figure 1t; Figure S1y). JA sensitivity assays showed that the root lengths of ospsal-ko exhibited markedly shorter while OsPsaL-ox showed more longer compared to the controls (Figure 1u,v; Figure S1z,a2), suggesting that the negative regulatory role of OsPsaL in the JA pathway. Collectively, we discovered a new susceptibility factor, OsPsaL, and demonstrated that knocking out the OsPsaL gene in rice enhanced both rice yield and antiviral immunity. Therefore, this study provides valuable genetic resources for future research on improving both rice yield and antiviral immunity.

大米 ( Oryza sativa ) 是全球一半以上人口的主食。包括病毒在内的各种植物病原体对水稻产量和质量构成重大威胁。南方水稻黑条矮缩病毒（SRBSDV）属于呼肠孤病毒科斐济病毒属，已成为导致亚洲国家农作物严重损失的主要病毒种类（Zhang et al ., 2023 ）。由于缺乏自然抗性，传统育种和商业水稻品种在实现病毒抗性方面面临挑战。因此，利用生物技术方法创建和培育抗性种质对于病毒性疾病的防治至关重要。

产氧光合作用是高等植物将阳光转化为化学能的主要过程。光合作用的光反应由光系统 I 和 II（PSI 和 PSII）驱动。 PSI 是一种膜蛋白复合物，作为光合作用机制的一个组成部分，能够实现阳光驱动的跨膜电子转移（Malavath等， 2018 ；Varotto等， 2000 ）。作为 PSI 的组成部分，PsaL 对于 PSI 三聚体的形成至关重要，这一过程可能依赖于钙离子与 PsaL 亚基的结合。然而，PsaL 在植物生长和免疫中的参与仍不清楚。

成簇规则间隔短回文重复序列/CRISPR相关9 (CRISPR/Cas9)技术已被有效利用，通过从头驯化从野生物种中创造出新品种（Bai等人， 2023 ）。在这项研究中，我们展示了 CRISPR/Cas9 在水稻中的成功应用，以创建具有优异农艺性状和对 SRBSDV 抗性的转基因品系。我们首先发现SRBSDV感染后OsPsaL基因的表达水平显着下调（图1a）。然后，我们在日本晴（NIP）背景下通过 CRISPR/Cas9 系统生成了两个独立的ospsal-ko突变体（ ospsal-1和ospsal-2 ）（图 1b，c）。随后，我们使用叶绿素荧光来评估转基因植物的光合特性。与野生型相比， ospsal-ko中的电子传输速率（ETR）和净光合效率（pN）显着增加，而 Y（NO）（散热和荧光失控的指标）随着光强度的增加而下降（图1d–f)，表明突变体的光合作用得到增强。我们进一步构建了过表达OsPsaL转基因水稻，命名为OsPsaL-ox （ OsPsaL-3#和OsPsaL-4# ）（图 S1a、b）。与野生型相比， OsPsaL-ox植物的电子传输速率和净光合效率降低，但 Y (NO) 没有变化（图 S1c-e）。 统计分析显示， ospsal-ko每株植物的分蘖数、圆锥花序和籽粒数量较多，但与野生型植物相比，它们在种子大小和千粒重或直链淀粉比例方面没有表现出差异（图1g-l） 。 OsPsaL-3的粒长和千粒重显着高于野生型植物（图S1f，g）。然而， OsPsaL过度表达的植物表现出显着减少的分蘖和圆锥花序（图 S1h，i）；因此，植物的总体产量明显降低（图S1j）。这些发现表明， ospsal-ko 的光合作用得到增强，产量也有所增加。

接下来，我们的目标是研究OsPsaL在 SRBSDV 感染中的作用。接种 SRBSDV 约 30 天后， ospsal-ko 的矮化程度低于对照（图 1m）。病毒含量检测表明，与对照相比，突变体ospsal-ko中病毒外壳蛋白P10和SRBSDV RNA（ S2 、 S4和S6 ）的含量显着降低（图1n，o）。而与野生型植物相比， OsPsaL-ox植物在 RNA 和蛋白质水平上表现出更严重的矮化和更高的病毒积累（图 S1l-n）。总之，这些结果表明OsPsaL在水稻抵抗 SRBSDV 的防御中发挥负面作用。为了探索ospsal-ko的广谱抗病性，我们用不同类型的水稻病毒（稻条病毒，RSV）接种转基因植物，结果表明ospsal-ko也表现出对RSV的抗性，而OsPsaL-ox表现出更高的抗性对 RSV 的敏感性（图 1p–r 和图 S1o–q）。

我们进一步对 ZH11 和OsPsaL-ox进行转录组测序以响应 SRBSDV 感染。检查了OsPsaL-3# -V 与OsPsaL-3 #-H 比较中具有特异表达但在 ZH11-V 与 ZH11-H 中未发现的差异表达基因，从而鉴定了 2178 个基因（图 S1r）。与 ZH11 相比，这些基因在 SRBSDV 感染的OsPsaL-3#植物中大部分受到抑制。 GO分析表明这些下调基因在光合作用中高度富集（图S1s）。这些发现表明， OsPsaL-ox水稻的光合作用受到 SRBSDV 的显着损害。此外，转录组的综合分析显示，与 ZH11 相比， OsPsaL-3 # 中几个茉莉酸 (JA) 相关基因显着下调（图 S1t）。 JA 被普遍认为是重要的抗病毒途径（Li等， 2021 ；Zhang等， 2023 ）。进一步的 RT-qPCR 检测表明，与野生型植物相比，JA 相关基因（ OsLOX2 、 OsAOC 、 OsAOS2和OsJMT1 ）的表达在ospsal-ko中显着激活，但在OsPsaL-ox中受到抑制（图 1s；图 S1u-x） ）。 JA 含量测定表明，与野生型植物相比， ospsal-ko中的 JA 浓度显着较高，但OsPsaL-ox中的 JA 浓度较低（图 1t；图 S1y）。 JA敏感性测定表明，与对照相比， ospsal-ko的根长明显较短，而OsPsaL-ox的根长更长（图1u，v；图S1z，a2），这表明OsPsaL在JA途径中的负调节作用。总的来说，我们发现了一种新的易感因子OsPsaL ，并证明敲除水稻中的OsPsaL基因可以提高水稻产量和抗病毒免疫力。因此，本研究为今后提高水稻产量和抗病毒免疫力的研究提供了宝贵的遗传资源。