![]() ![]() Proline-rich proteins (PRPs) are involved in cell-wall signaling, plant development, and stress responses ( Kavi Kishor et al., 2015). In particular, they have been successfully applied to tomatoes for drought tolerance ( Wang et al., 2017a Li et al., 2019), salt tolerance ( Tran et al., 2020 Vu et al., 2020b Wang et al., 2021), and chilling and heat tolerance ( Li et al., 2018a Yin et al., 2018). Moreover, CRISPR-Cas9 or/and CRISPR-Cas12a were employed to effectively edit genes for obtaining enhanced abiotic stress tolerance in various plant species such as rice and wheat ( Shan et al., 2013 Shan et al., 2014 Endo et al., 2016 Osakabe et al., 2018), tomato ( Brooks et al., 2014 Tran et al., 2020 Vu et al., 2020b), and apple ( Malnoy et al., 2016 Nishitani et al., 2016 Osakabe et al., 2018 Charrier et al., 2019). CRISPR-Cas-based targeted mutants of CLAVATA/WUS showed different ranges in tomato shoot meristem size ( Fletcher, 2018). Subsequently, stable inheritance of the edited alleles in the next generations was observed through genotyping and phenotyping ( Brooks et al., 2014). The application of CRISPR-Cas9 in tomato genome engineering obtained targeted mutations at four different loci. Therefore, new tomato cultivars capable of biotic and abiotic stress tolerance are important subjects of breeding using GE technology. Recently, unforeseen climate changes have caused many biotic and abiotic stresses that negatively affect multiple aspects of tomato production, such as yield and quality, thereby threatening agriculture sustainability. It has been successful in modifying tomato genomes and precision breeding to create new alleles, not including linkage drag ( Zsögön et al., 2017 Li et al., 2018b Zsögön et al., 2018 Vu et al., 2020a). CRISPR-Cas-based GE has been effectively applied for plant breeding at high precision levels ( Chen et al., 2019). Genome editing (GE) technology using CRISPR-Cas nucleases has recently emerged as a revolutionary plant breeding technology and shows promise to the agriculture industry ( Belhaj et al., 2013 Chen et al., 2019 Vu et al., 2022). CRISPR-Cas9-based precise domain editing of the SlHyPRP1 gene generated multi-stress-tolerant alleles that could be used as genetic materials for tomato breeding. However, the edited alleles enhanced susceptibility against Fusarium oxysporum f. tomato ( Pto) DC3000, the growth of the bacterium was significantly reduced by 2.0- to 2.5-fold compared to that in WT plants. When the edited lines were challenged with pathogenic bacteria of Pseudomonas syringae pv. Moreover, the PR1v1 line continuously grew after 5 days of water cutoff. Similarly, plants carrying either the domains of the PRD removal variant (PR1v1) or 8CM removal variants (PR2v2 and PR2v3) showed better germination under osmosis stress (up to 200 mM mannitol) compared to the WT control. Our data reveal that the 8CM removal variants of HK and the KO alleles of both HK and 15T01 cultivars exhibited moderate heat stress tolerance. In this study, we characterized the edited lines under several abiotic and biotic stresses to examine the possibility of multiple stress tolerance. We subsequently demonstrated that eliminating the PRD domain of HyPRP1 in tomatoes conferred the highest level of salinity tolerance. In our previous report, a tomato gene encoding hybrid proline-rich protein 1 (HyPRP1), a negative regulator of salt stress responses, has been edited using a CRISPR-Cas9 multiplexing approach that resulted in precise eliminations of its functional domains, proline-rich domain (PRD) and eight cysteine-motif (8CM). Recently, CRISPR-Cas9-based genome editing has been widely used for plant breeding. 6Nulla Bio R&D Center, Nulla Bio Inc., Jinju, Republic of Korea.5National Key Laboratory for Plant Cell Biotechnology, Agricultural Genetics Institute, Hanoi, Vietnam.4Division of Life Science, Gyeongsang National University, Jinju, Republic of Korea.3Division of Horticultural Science, Gyeongsang National University, Jinju, Republic of Korea.2Crop Science and Rural Development Division, College of Agriculture, Bac Lieu University, Bac Lieu, Vietnam. ![]() 1Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju, Republic of Korea.Mil Thi Tran 1,2,3†, Geon Hui Son 1†, Young Jong Song 1†, Ngan Thi Nguyen 1, Seonyeong Park 1, Thanh Vu Thach 1, Jihae Kim 1, Yeon Woo Sung 1, Swati Das 1, Dibyajyoti Pramanik 1, Jinsu Lee 1, Ki-Ho Son 3, Sang Hee Kim 1,4*‡, Tien Van Vu 1,5*‡ and Jae-Yean Kim 1,4,6*‡ ![]()
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