Abstract
Virus-induced gene silencing (VIGS) has been employed for the high-throughput analysis of endogenous gene function. We developed a CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) for the efficient generation of viral transcripts in plants. Using the novel pCF93 vector, we identified genes related to male sterility in watermelon (Citrullus lanatus), which is recalcitrant to genetic transformation. We previously reported reference-based and de novo transcriptomic profiling for the detection of differentially expressed genes between a male fertile line (DAH3615) and its near isogenic male sterile line (DAH3615-MS). Based on the RNA-seq results, we identified 38 de novo-exclusive differentially expressed genes (DEDEGs) that are potentially responsible for male sterility. Partial genes of 200∼300bp were cloned into pCF93 which was then inoculated into DAH, a small type of watermelon that enables high-throughput screening with a small cultivation area. In this manner, we simultaneously characterized phenotypes associated with the 38 candidate genes in a common-sized greenhouse. Eight out of the 38 gene-silenced plants produced male sterile flowers with abnormal stamens and no pollens. Gene expression levels in flowers were validated via RT-qPCR. Stamen histological sections from male sterile floral buds and mature flowers showed developmental disruption and shrunken pollen sacs. Based on the current findings, we believe that the novel pCF93 vector and our VIGS system facilitate high-throughput analysis for the study of gene function in watermelons.
One sentence summary The CaMV 35S promoter-driven cucumber fruit mottle mosaic virus-Cm vector (pCF93) facilitates large-scale validation of male sterility-related gene functions in watermelon.
Footnotes
↵* Author for communication: gplee{at}cau.ac.kr
↵† Senior author
GL and SR designed the study, SR performed the experiments, analyzed the data, and drafted the manuscript. YJ performed the cloning and histological analysis. JP performed the Agroinoculation and phenotyping. GL supervised the data analysis, revised the manuscript, and funded the project. All authors reviewed and approved the submitted version of the manuscript.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (https://academic.oup.com/plphys/pages/general-instructions) is Gung Pyo Lee (gplee{at}cau.ac.kr).
[Email address of Author for Contact] Sun-Ju Rhee (SR): sjrhee825{at}gmail.com
Yoon Jeong Jang (YJ): jangyj770{at}gmail.com
Jun-Young Park (JP): parkjy1218{at}naver.com
Gung Pyo Lee (GL): gplee{at}cau.ac.kr