Identification and Functional Analysis of Key Factors Determining the Different Pathogenicity of Two Tomato Leaf Curl New Delhi Virus Isolates in Cucurbitaceous Plants

Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus (family Geminiviridae) originally isolated from tomatoes and later evolved to cross-infect cucurbit crops, causing severe economic damage in Asia and Europe. In this study, we sequenced and characterized complete genomes of two ToLCNDV isolates collected from Hebei (ToLCNDV-HB) and Jiangsu (ToLCNDV-JS) provinces of China infecting melon. We constructed infectious clones for ToLCNDV-HB and ToLCNDV-JS, which could systemically infect Nicotiana benthamiana, tomato, and four species of cucurbitaceous plants. Notably, ToLCNDV-HB induced more severe symptoms and accumulated higher viral DNA and protein accumulation than ToLCNDV-JS in N. benthamiana, melon, and bottle gourd. Sequence analysis showed that sequence variations are present only in AV2, AC1, and AC4. However, only the AV2 ORF from ToLCNDV-HB was more efficient than that from that ToLCNDV-JS in enhancing potato X virus’s pathogenicity and suppressing post-transcriptional gene silencing (PTGS). An AV2-swapping experiment between ToLCNDV-HB and ToLCNDV-JS confirmed its vital role in determining the differential pathogenicity. Further evidence shows that virions from both clones are mechanically transmissible. This is the first report comparing the differential pathogenicity of two Chinese ToLCNDV isolates in cucurbits. The AV2 protein, a key pathogenicity determinant, represents a potential target for breeding ToLCNDV-resistant cucurbit varieties.