Sweet potato (Ipomoea batatas) is one of the most important crops in the world, and its production rate is mainly decreased by the sweet potato virus disease (SPVD) caused by the co‐infection of sweet potato chlorotic stunt virus (SPCSV) and sweet potato feathery mottle virus. However, methods for improving SPVD resistance have not been established. Thus, this study aimed to enhance SPVD resistance by targeting one of its important pathogenesis‐related factors (i.e., SPCSV‐RNase3) by using the CRISPR‐Cas13 technique. First, the RNA targeting activity of four CRISPR‐Cas13 variants were compared using a transient expression system in Nicotiana benthamiana. LwaCas13a and RfxCas13d had more efficient RNA and RNA virus targeting activity than PspCas13b and LshCas13a. Driven by the pCmYLCV promoter for the expression of gRNAs, RfxCas13d exhibited higher RNA targeting activity than that driven by the pAtU6 promoter. Furthermore, the targeting of SPCSV‐RNase3 using the LwaCas13a system inhibited its RNA silencing suppressor activity and recovered the RNA silencing activity in N. benthamiana leaf cells. Compared with the wild type, transgenic N. benthamiana plants carrying an RNase3‐targeted LwaCas13a system exhibited enhanced resistance against turnip mosaic virus TuMV‐GFP and cucumber mosaic virus CMV‐RNase3 co‐infection. Moreover, transgenic sweet potato plants carrying an RNase3‐targeted RfxCas13d system exhibited substantially improved SPVD resistance. This method may contribute to the development of SPVD immune germplasm and the enhancement of sweet potato production in SPVD‐prevalent regions.

Phosphatidylserine synthase (PSS)-mediated phosphatidylserine (PS) synthesis is crucial for plant development. However, little is known about the contribution of PSS to Na⁺homeostasis regulation and salt tolerance in plants. Here, we cloned theIbPSS1gene, which encodes an ortholog ofArabidopsis AtPSS1, from sweet potato (Ipomoea batatas(L.) Lam.). The transient expression ofIbPSS1inNicotiana benthamianaleaves increased PS abundance. We then established an efficientAgrobacterium rhizogenes-mediated in vivo root transgenic system for sweet potato. Overexpression ofIbPSS1through this system markedly decreased cellular Na⁺accumulation in salinized transgenic roots (TRs) compared with adventitious roots. The overexpression ofIbPSS1enhanced salt-induced Na⁺/H⁺antiport activity and increased plasma membrane (PM) Ca²⁺-permeable channel sensitivity to NaCl and H₂O₂in the TRs. We confirmed the important role ofIbPSS1in improving salt tolerance in transgenic sweet potato lines obtained from anAgrobacterium tumefaciens-mediated transformation system. Similarly, compared with the wild-type (WT) plants, the transgenic lines presented decreased Na⁺accumulation, enhanced Na⁺exclusion, and increased PM Ca²⁺-permeable channel sensitivity to NaCl and H₂O₂in the roots. Exogenous application of lysophosphatidylserine triggered similar shifts in Na⁺accumulation and Na⁺and Ca²⁺fluxes in the salinized roots of WT. Overall, this study provides an efficient and reliable transgenic method for functional genomic studies of sweet potato. Our results revealed thatIbPSS1contributes to the salt tolerance of sweet potato by enabling Na⁺homeostasis and Na⁺exclusion in the roots, and the latter process is possibly controlled by PS reinforcing Ca²⁺signaling in the roots.

Sweetpotato (Ipomoea batatas L.) is a strategic crop with both economic and energy value. However, improving sweetpotato varieties through traditional breeding approaches can be a time-consuming and labor-intensive process due to the complex genetic nature of sweetpotato as a hexaploid species (2n = 6x = 90). Double haploid (DH) breeding, based on in vivo haploid induction, provides a new approach for rapid breeding of crops. The success of haploid induction can be achieved by manipulating specific genes. Two of the most critical genes, DMP (DUF679 membrane proteins) and MTL (MATRILINEAL), have been shown to induce haploid production in several species. Here, we identified and characterized DMP and MTL genes in sweetpotato using gene family analysis. In this study, we identified 5 IbDMPs and 25 IbpPLAs. IbDMP5 and IbPLAIIs (IbPLAIIκ, IbPLAIIλ, and IbPLAIIμ) were identified as potential haploid induction (HI) genes in sweetpotato. These results provide valuable information for the identification and potential function of HI genes in sweetpotato and provide ideas for the breeding of DH lines.