Agrobacterium rhizogenes-mediated marker-free transformation and gene editing system revealed that AeCBL3 mediates the formation of calcium oxalate crystal in kiwifruit.

The transformation and gene editing of the woody species kiwifruit are difficult and time-consuming. The fast and marker-free genetic modification system for kiwifruit has not been developed yet. Here, we establish a rapid and efficient marker-free transformation and gene editing system mediated by Agrobacterium rhizogenes for kiwifruit. Moreover, a removing-root-tip method was developed to significantly increase the regeneration efficiency of transgenic hairy roots. Through A. rhizogenes-mediated CRISPR/Cas9 gene editing, the editing efficiencies of CEN4 and AeCBL3 achieved 55 and 50%, respectively. And several homozygous knockout lines for both genes were obtained. Our method has been successfully applied in the transformation of two different species of kiwifruit (Actinidia chinensis 'Hongyang' and A.eriantha 'White'). Next, we used the method to study the formation of calcium oxalate (CaOx) crystals in kiwifruit. To date, little is known about how CaOx crystal is formed in plants. Our results indicated that AeCBL3 overexpression enhanced CaOx crystal formation, but its knockout via CRISPR/Cas9 significantly impaired crystal formation in kiwifruit. Together, we developed a fast maker-free transformation and highly efficient CRISPR-Cas9 gene editing system for kiwifruit. Moreover, our work revealed a novel gene mediating CaOx crystal formation and provided a clue to elaborate the underlying mechanisms.

Genome-Wide Identification of HSF Gene Family in Kiwifruit and the Function of AeHSFA2b in Salt Tolerance.

Heat shock transcription factors (HSFs) play a crucial role in regulating plant growth and response to various abiotic stresses. In this study, we conducted a comprehensive analysis of the AeHSF gene family at genome-wide level in kiwifruit (Actinidia eriantha), focusing on their functions in the response to abiotic stresses. A total of 41 AeHSF genes were identified and categorized into three primary groups, namely, HSFA, HSFB, and HSFC. Further transcriptome analysis revealed that the expression of AeHSFA2b/2c and AeHSFB1c/1d/2c/3b was strongly induced by salt, which was confirmed by qRT-PCR assays. The overexpression of AeHSFA2b in Arabidopsis significantly improved the tolerance to salt stress by increasing AtRS5, AtGolS1 and AtGolS2 expression. Furthermore, yeast one-hybrid, dual-luciferase, and electrophoretic mobility shift assays demonstrated that AeHSFA2b could bind to the AeRFS4 promoter directly. Therefore, we speculated that AeHSFA2b may activate AeRFS4 expression by directly binding its promoter to enhance the kiwifruit's tolerance to salt stress. These results will provide a new insight into the evolutionary and functional mechanisms of AeHSF genes in kiwifruit.