Rice (Oryza sativa) is one of the most important staple foods for more than half of the world's population (Chen et al. 2011). However, many viral diseases severely challenge rice production in many global areas, such as Southern rice black-streaked dwarf virus (SRBSDV), which is severely epidemic and has caused 30-50% rice yield losses in southern China and Southeast Asia in the last decade (Alonso et al. 2019). Although SRBSDV has been successfully controlled by international cooperation, it still exists in the majority of rice producing areas of eastern China, with periodic outbreaks in a few rice production areas and the potential for additional widespread outbreaks (Zhou et al. 2013). One of the most effective strategies to prevent viral diseases is growing resistant or tolerant varieties; nevertheless, almost all cultivated rice varieties are susceptible to SRBSDV (Wang et al. 2017; Yu et al. 2017; Zhou et al. 2021). CRISPR/Cas-based genome editing is an alternative method for accelerating rice improvement (Ma et al. 2015). The availability of rice reference genome sequences and the CRISPR/Cas9-editing system has made it possible to develop disease-resistant or disease-tolerant rice by precisely editing endogenous genes (Zhao et al. 2020).
Vacuolar ATPases (v-ATPases) are proton pumps for proton translocation across membranes that utilize energy derived from ATP hydrolysis (Forgac et al. 2007; Mazhab-Jafari et al. 2017). Eukaryotic v-ATPases are multiprotein complexes, and v-ATPase subunit d (v-ATPase d) is part of an integral, membrane-embedded V0 complex (Hohlweg et al. 2018). The pathogens Sarocladium oryzae and Pseudomonas fuscovaginae cause rice sheath rot and produce cyclic lipopeptides to inhibit membrane-bound H+-ATPase pumps in the rice plant, resulting in reduced abscisic acid (ABA), jasmonate acid (JA) and auxin levels and grain yield in rice (Peeters et al. 2020). Plant hormones are pivotal for biotic and abiotic resistance, and rice hormones have diverse functions in rice resistance against different viruses (Xie et al. 2018; Yan et al. 2015; Zhang et al. 2020). Therefore, Osv-ATPase d could be an alternative target for gene editing by CRISP/Cas9 to enhance viral resistance in rice.
In this study, CRISPR/Cas9-based genome-editing technology was employed to edit Osv-ATPase d in Nipponbare (Oryza sativa L. cv. Japonica, NIP), which is highly susceptible to SRBSDV and Rice stripe virus (RSV) (Zhang et al. 2019). Two guide RNAs were designed to target the first exon of Osv-ATPase d by CRISPR Design (http://cripsr.mit.edu) (Fig. 1a). Specific single guide RNAs (sgRNAs) targeted to Osv-ATPase d were selected and constructed by universal primers (Table S1) , which was used to transform the rice cultivar NIP by the Agrobacterium mediated transformation as previously described (Ma et al., 2015). Five independent T0 lines were found to carry heterozygous mutations of Osv-ATPase d. From the T1 segregation population, a CAS9-free homozygous mutant with knockout of Osv-ATPase d (hereafter named line 5) was identified. Conventional Sanger sequencing verified that a “C” insertion resulted in a frameshift mutant with a “G” deletion in Osv-ATPase d (Figure 1a). This plant and its offspring were selected and used for further trait analysis.
The growth trial of editing line 5 grown in pots under greenhouse conditions showed normal growth with no morphological differences when compared to wild-type plants at 50 days of age (Figure 1b). No adverse effect was observed regarding spike morphology (Figure 1c) or the yield characteristics of spike length, number of spikelets, grain number per spike and 1000-grain weight between editing line 5 and wild-type plants (Table 1). These results suggested that there was no detrimental impact of knocking out Osv-ATPase d in rice.
Table 1 Agronomic traits of the line 5 and the wild type
Sample
|
Spike length (cm)
|
Number of spikelets
|
grain number per spike
|
1000-grain weight (g)
|
line 5
|
20.26±0.56a
|
9.67±0.67a
|
114.33±1.45a
|
19.76±1.18a
|
NIP
|
19.37±0.47a
|
9.67±0.33a
|
104.00±4.04a
|
20.91±0.91a
|
Notes: Letters indicate significantly different values using Student’s t test (ρ < 0.05).
To gain insight into the functional profiles of Osv-ATPase d in rice, the transcriptomic response (dataset was permanently deposited in GenBank at accession number: PRJNA753714) of editing line 5 plants was comparatively analyzed with that of wild-type plants. Compared with wild-type plants, a total of 664 differentially expressed genes (DEGs) were induced in the editing line 5 seedlings (15 days old) (Fig 2a). KEGG pathways with enrichment of 443 significantly upregulated and 221 significantly downregulated genes revealed that several metabolic pathways were altered between editing line 5 and the wild type, such as phenylpropanoid biosynthesis, plant hormone signal transduction and the MAPK signalling pathway (Fig 2b). These findings showed that Osv-ATPase d is probably involved in mediating the biosynthesis of plant hormones and resistance to pathogens of rice and may be involved in the molecular mechanisms of both pathways.
Transcriptomic analysis showed that Osv-ATPase d is involved in plant hormone mediation; thus, the plant hormones in editing line 5 and the wild type were then quantified by ultra-high-performance liquid chromatography-triple quadrupole mass spectrometry (UPLC-MS/MS) (Hu et al. 2020; Peeters et al. 2020). As expected, Osv-ATPase d was indeed involved in mediating plant hormone biosynthesis. Compared with wild-type plants, editing line 5 showed significantly increased JA and ABA biosynthesis (Fig 2c and 2d), but there was no effect on the biosynthesis of five other plant hormones, including 1-aminocyclopropanecarboxylic acid (ACC), indoleacetic acid (IAA), salicylic acid (SA) and trans-zeatin (tZ) (Table S2).
Transcriptomic and plant hormone biosynthesis analysis showed that Osv-ATPase d may mediate resistance in rice. Three replicates of editing line 5 were evaluated for resistance against SRBSDV and RSV. SRBSDV disease symptom observations showed that at 30 dpi, the NIP plants showed more severe stunting upon SRBSDV infection (Fig 3a), and the SRBSDV disease incidence (Fig 3b) and accumulation of SRBSDV virions (Fig 3c) in the wild-type plants were significantly higher than those in the line 5 plants. In contrast, the editing line 5 plants displayed higher susceptibility to RSV than the wild-type plants (Fig 3d-3f). Further analyses showed that editing line 5 in rice had no significant effect on virus-transmitting vector infestation. These results indicated that Osv-ATPase d can differentially regulate rice resistance to SRBSDV and RSV infection.
Altogether, the Osv-ATPase d knockout mutant of rice showed different levels of resistance to important viruses, SRBSDV and RSV, and did not show any detrimental effect of gene knockout on plant growth or yield productivity. Osv-ATPase d can be selected as a potential target for resistance breeding in rice.