Development and Analysis of Infectious and Fluorescent Clones of Cotton Leafroll Dwarf Virus-AL

Cotton leafroll dwarf virus (CLRDV) is a plant viral pathogen first reported in the United States in 2017 that causes yield loss in certain cotton ( Gossypium hirsutum L) cultivars but is asymptomatic in others. In Argentina, an infectious clone of the typical and atypical strains of CLRDV were developed to facilitate biological studies and aid in the development of resistant cotton cultivars. Two infectious clones derived from the original Alabama isolate (Macon 1) were created using Agrobacterium tumefaciens for plant infection. The first one, CLRDV-Macon1 (CLRDV-Ma1) was constructed by inserting the full-length sequence of CLRDV into the pJL89 plasmid, followed by transformation of A. tumefaciens strain LBA4404. This clone systemically infects cotton and Nicotiana benthamiana and was detectable in transfected plants by PCR amplification. A second clone, CLRDV-M-GFP, was constructed by replacing a fragment of the open reading frame 3-5 region of CLRDV-Ma1 with enhanced green fluorescent protein (EGFP). This clone expresses EGFP, making visualization possible by fluorescence microscopy in infiltrated cotton cotyledons, and is detectable using commercial antibodies. However, during the infection cycle, EGFP was found to be truncated, resulting from unintended mutations. This study reports the first infectious clones of a U.S. isolate of CLRDV and contributes new knowledge about the biology of poleroviruses. [Formula: see text] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .