NSvc4 Encoded by Rice Stripe Virus Targets Host Chloroplasts to Suppress Chloroplast-Mediated Defense

Our previous research found that NSvc4, the movement protein of rice stripe virus (RSV), could localize to the actin filaments, endoplasmic reticulum, plasmodesmata, and chloroplast, but the roles of NSvc4 played in the chloroplast were opaque. Here, we confirm the accumulation of NSvc4 in the chloroplasts and the N-terminal 1–73 amino acids of NSvc4 are sufficient to localize to chloroplasts. We provide evidence to show that chloroplast-localized NSvc4 can impair the chloroplast-mediated immunity. Expressing NSvc4 in Nicotiana benthamiana leaves results in the decreased expression of defense-related genes NbPR1, NbPR2, and NbWRKY12 and the inhibition of chloroplast-derived ROS production. In addition, generation of an infectious clone of potato virus X (PVX) carrying NSvc4 facilitates PVX infection in N. benthamiana plants. Moreover, we identify two chloroplast-related host factors, named NbGAPDH-A and NbPsbQ1, both of which can interact with NSvc4. Knockdown of NbGAPDH-A or NbPsbQ1 can both promote RSV infection. Our results decipher a detailed function of NSvc4 in the chloroplast.

Spontaneous Mutation in the Movement Protein of Citrus Leprosis Virus C2, in a Heterologous Virus Infection Context, Increases Cell-to-Cell Transport and Generates Fitness Advantage

Previous results using a movement defective alfalfa mosaic virus (AMV) vector revealed that citrus leprosis virus C (CiLV-C) movement protein (MP) generates a more efficient local movement, but not more systemic transport, than citrus leprosis virus C2 (CiLV-C2) MP, MPs belonging to two important viruses for the citrus industry. Here, competition experiment assays in transgenic tobacco plants (P12) between transcripts of AMV constructs expressing the cilevirus MPs, followed by several biological passages, showed the prevalence of the AMV construct carrying the CiLV-C2 MP. The analysis of AMV RNA 3 progeny recovered from P12 plant at the second viral passage revealed the presence of a mix of progeny encompassing the CiLV-C2 MP wild type (MPWT) and two variants carrying serines instead phenylalanines at positions 72 (MPS72F) or 259 (MPS259F), respectively. We evaluated the effects of each modified residue in virus replication, and cell-to-cell and long-distance movements. Results indicated that phenylalanine at position 259 favors viral cell-to-cell transport with an improvement in viral fitness, but has no effect on viral replication, whereas mutation at position 72 (MPS72F) has a penalty in the viral fitness. Our findings indicate that the prevalence of a viral population may be correlated with its greater efficiency in cell-to-cell and systemic movements.

Suppression of a dsRNA-induced plant immunity pathway by viral movement protein

The virome of plants is dominated by RNA viruses and several of these cause devastating diseases in cultivated plants leading to global crop losses. To infect plants, RNA viruses engage in complex interactions with compatible plant hosts. In cells at the spreading infection front, RNA viruses replicate their genome through double-stranded RNA (dsRNA) intermediates and interact with cellular transport processes to achieve cell-to-cell movement of replicated genome copies through cell wall channels called plasmodesmata (PD). In order to propagate, viruses also must overcome host defense responses. In addition to triggering the antiviral RNA silencing response, RNA virus infection also elicits pattern-triggered immunity (PTI) whereby dsRNA, a hallmark of virus replication, acts as an important elicitor. This innate antiviral immune response is also triggered when dsRNA is applied externally and does not require sequence homology to the virus. However, the mechanism by which PTI restricts virus infection is not known. Here, we show that dsRNA inhibits the progression of virus infection by triggering callose deposition at plasmodesmata and the inhibition of transport through these cell-to-cell communication channels. The dsRNA-induced signaling pathway leading to callose deposition is independent of ROS production and thus distinguished from pathways triggered by bacterial and fungal elicitors. The dsRNA-induced host response at plasmodesmata is suppressed by the Tobacco mosaic virus movement protein (MP). Thus, the virus uses MP to inhibit innate dsRNA-induced immunity at plasmodesmata, which could be a general strategy of phytoviruses to overcome plant defenses and spread infection.

Autophagy Inhibits Intercellular Transport of Citrus Leaf Blotch Virus by Targeting Viral Movement Protein

Autophagy is an evolutionarily conserved cellular-degradation mechanism implicated in antiviral defense in plants. Studies have shown that autophagy suppresses virus accumulation in cells; however, it has not been reported to specifically inhibit viral spread in plants. This study demonstrated that infection with citrus leaf blotch virus (CLBV; genus Citrivirus, family Betaflexiviridae) activated autophagy in Nicotiana benthamiana plants as indicated by the increase of autophagosome formation. Impairment of autophagy through silencing of N. benthamiana autophagy-related gene 5 (NbATG5) and NbATG7 enhanced cell-to-cell and systemic movement of CLBV; however, it did not affect CLBV accumulation when the systemic infection had been fully established. Treatment using an autophagy inhibitor or silencing of NbATG5 and NbATG7 revealed that transiently expressed movement protein (MP), but not coat protein, of CLBV was targeted by selective autophagy for degradation. Moreover, we identified that CLBV MP directly interacted with NbATG8C1 and NbATG8i, the isoforms of autophagy-related protein 8 (ATG8), which are key factors that usually bind cargo receptors for selective autophagy. Our results present a novel example in which autophagy specifically targets a viral MP to limit the intercellular spread of the virus in plants.

Phase separation of a plant virus movement protein and cellular factors support virus-host interactions

Both cellular and viral proteins can undergo phase separation and form membraneless compartments that concentrate biomolecules. The p26 movement protein from single-stranded, positive-sense Pea enation mosaic virus 2 (PEMV2) separates into a dense phase in nucleoli where p26 and related orthologues must interact with fibrillarin (Fib2) as a pre-requisite for systemic virus movement. Using in vitro assays, viral ribonucleoprotein complexes containing p26, Fib2, and PEMV2 genomic RNAs formed droplets that may provide the basis for self-assembly in planta. Mutating basic p26 residues (R/K-G) blocked droplet formation and partitioning into Fib2 droplets or the nucleolus and prevented systemic movement of a Tobacco mosaic virus (TMV) vector in Nicotiana benthamiana. Mutating acidic residues (D/E-G) reduced droplet formation in vitro, increased nucleolar retention 6.5-fold, and prevented systemic movement of TMV, thus demonstrating that p26 requires electrostatic interactions for droplet formation and charged residues are critical for nucleolar trafficking and virus movement. p26 readily partitioned into stress granules (SGs), which are membraneless compartments that assemble by clustering of the RNA binding protein G3BP following stress. G3BP is upregulated during PEMV2 infection and over-expression of G3BP restricted PEMV2 RNA accumulation >20-fold. Deletion of the NTF2 domain that is required for G3BP condensation restored PEMV2 RNA accumulation >4-fold, demonstrating that phase separation enhances G3BP antiviral activity. These results indicate that p26 partitions into membraneless compartments with either proviral (Fib2) or antiviral (G3BP) factors.