Identification of the Matrix Protein as a Conserved and Central Determinant of Superinfection Exclusion in Plant Rhabdoviruses

Superinfection exclusion (SIE) is a finely tuned virus-virus interaction mechanism closely linked to the viral infection cycle. However, the mechanistic basis of SIE remains incompletely understood in plant viruses, particularly among negative-sense, single-stranded RNA viruses. In this study, we first describe the development of an efficient reverse genetics system for the plant nucleorhabdovirus Physostegia chlorotic mottle virus (PhCMoV) by codon optimisation of the large polymerase coding sequence. Using fluorescently tagged variants of PhCMoV, as well as three additional closely or distantly related plant rhabdoviruses, we found that each rhabdovirus displayed homotypic SIE. Moreover, two closely related alphanucleorhabdoviruses, PhCMoV and eggplant mottled dwarf virus, also exhibited mutual exclusion. Loss- and gain-of-function reverse genetics analyses identified the rhabdovirus matrix (M) protein as the central SIE effector: M-deficient mutant viruses lost exclusion capacity, whereas ectopically expressed heterologous M proteins conferred SIE against otherwise compatible, distantly related rhabdoviruses. Additional functional assays demonstrated that the ability of rhabdovirus M proteins to suppress cognate and noncognate viral RNA synthesis correlated with the intra- and interspecies SIE capacity. The widespread occurrence of SIE across distinct plant rhabdoviruses underscores its importance for understanding the viral replication cycle and highlights its practical relevance for the development of novel virus control strategies.