Chloroplast localization of citrus psorosis virus movement protein is essential to exert its viral movement activity

MARCHESINI ANA; BORNIEGO, MARIA BELÉN; ROBLES LUNA, GABRIEL; PEÑA, EDUARDO JOSÉ

Mendoza

Congreso; SAIB LVIII; 2022

SAIB

Plant viral infections are responsible of huge crop losses and given their adaptability against defenses strategies, their eradication seems impossible. Understanding virus infection cycle and the interaction with host cellular machinery becomes essential to design protection strategies to ensure productivity and quality of vegetable products.Citrus psorosis virus (CPsV), the type member of Ophioviruses is the causal agent of Citrus psorosis, a worldwide distributed disease that affects all citrus species and is responsible for the death of 5% of productive plants per year. Early during infection CPsV causes chlorotic elongated spots in young leaves and progresses with the accumulation of gum in the vascular system, affecting plant yield and eventually causing bark scaling and plant dead. Natural resistance against CPsV has not been reported.CPsV is a three-partite negative sense RNA virus. RNA2 encodes a protein of 54 kDa (54K) that contains an aspartic protease domain and can self-cleave producing a 20 kDa protein at the C-terminal part that maintains protease activity, and a 34 kDa protein (movement protein, MP) at the N-terminal part. MP localizes almost exclusively in chloroplasts (Chl) and plasmodesmata (PD) and alters PD structures, increasing size exclusion limit (SEL) and forming tubules that facilitate cell-to-cell virus movement.Chloroplasts play a central role in photosynthesis and are involved in the synthesis plant defense and signaling hormones, which highlight the importance of this organelle in pathogen interaction and immune response coordination. Most plant viruses cause chlorosis early during infections, this is produced by alterations at chloroplasts and the consequent affection of photosynthetic rate. Transmission electron microscopy observations in herbaceous hosts infected with CPsV show modifications in both the structure and size as well as in cell chloroplasts arrangement compared to non infected plants. In silico analysis of the MP from CPsV and other ophioviruses identify a conserved chloroplast transit peptide (cTP). Together with MP and 54K localization, all this results suggest that CPsV targets the organelle as part of their infection cycle.To test the hypothesis, we designed 54K and MP mutants at the cTP as well as chimeras swapping cTPs with known chloroplast located proteins, such us Nicotiana benthamiana Rubisco small subunit (RbsS). These mutants were used to evaluate the role of chloroplast targeting in MP subcellular distribution, PD SEL regulation, virus cell-to-cell movement and chloroplast-mediated immune responses. Our results show that deleting native cTP from MP delays dramatically virus movement. Using a strong cTP (RbsS) which directs MP exclusively to chloroplast have a similar effect, which indicates that MP needs a dual localization to function properly. Further work will allow to mechanistically describe the specific role of MP at chloroplast.