Editing of OsPsaL gene improves both yield and antiviral immunity in rice

Abstract

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.

Figure 1

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Identification and functional validation of OsPsaL in the regulation of SRBSDV resistance. (a) The relative expression levels of OsPsaL gene after infection by SRBSDV. (b) The mutant type of ospsal-ko. (c, g–l) Phenotype of ospsal-ko (n = 10). Scale bar = 10 cm in (c) and Scale bar = 1 cm in (h). (d) Light response curves of ETR. (e)The index of Y(NO). (f) Net photosynthetic efficiency (pN). (m and p) Symptoms on wild-type and ospsal-ko plants inoculation with SRBSDV or RSV. Scale bars = 6 cm (Above); Scale bars = 1 cm (Below). (n and q) The accumulation of SRBSDV CP or RSV CP protein in infected NIP and ospsal-ko by western blot. RbcL serves as the loading control. (o, r) Relative expression levels of SRBSDV RNAs or RSV-CP in infected NIP and ospsal-ko. (s) Relative expression levels of JA pathway genes in ospsal-ko compared with NIP. OsUBQ5 was used as the internal reference gene. (t) The JA contents of NIP and ospsal-ko. (u) Phenotypes of ospsal-ko grown on different concentrations of MeJA for 7 days (n = 15), Scale bar = 2 cm. (v) Root lengths of ospsal-ko and NIP seedlings. All data are presented as means±SE, and statistical differences were determined using one-way ANOVA followed by Tukey's test (*P < 0.05).

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.