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.

Expression, purification and crystallization of the N-terminal Solanaceae domain of the Sw-5b NLR immune receptor

Plant nucleotide-binding domain and leucine-rich repeat receptors (NLRs) play crucial roles in recognizing pathogen effectors and activating plant immunity. The tomato NLR Sw-5b is a coiled-coil NLR (CC-NLR) immune receptor that confers resistance against tospoviruses, which cause serious economic losses in agronomic crops worldwide. Compared with other CC-NLRs, Sw-5b possesses an extended N-terminal Solanaceae domain (SD). The SD of Sw-5b is critical for recognition of the tospovirus viral movement protein NSm. An SD is also frequently detected in many NLRs from Solanaceae plants. However, no sequences homologous to the SD have been detected in animals or in plants other than Solanaceae. The properties of the SD protein are largely unknown, and thus 3D structural information is vital in order to better understand its role in pathogen perception and the activation of immune receptors. Here, the expression, purification and crystallization of Sw-5b SD (amino acids 1–245) are reported. Native and selenomethionine-substituted crystals of the SD protein belonged to space group P3112, with unit-cell parameters a = 81.53, b = 81.53, c = 98.44 Å and a = 81.63, b = 81.63, c = 98.80 Å, respectively. This is the first report of a structural study of the noncanonical SD domain of the NLR proteins from Solanaceae plants.

From a Movement-Deficient Grapevine Fanleaf Virus to the Identification of a New Viral Determinant of Nematode Transmission

Grapevine fanleaf virus (GFLV) and arabis mosaic virus (ArMV) are nepoviruses responsible for grapevine degeneration. They are specifically transmitted from grapevine to grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two viruses, were previously defined and exchanged to test their involvement in virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in virus transmission, thus representing a new viral determinant of nematode transmission.

Experimental and bioinformatic evidence that raspberry leaf blotch emaravirus P4 is a movement protein of the 30K superfamily

Emaravirus is a recently described genus of negative-strand RNA plant viruses. Emaravirus P4 protein localizes to plasmodesmata, suggesting that it could be a viral movement protein (MP). In the current study, we showed that the P4 protein of raspberry leaf blotch emaravirus (RLBV) rescued the cell-to-cell movement of a defective potato virus X (PVX) that had a deletion mutation in the triple gene block 1 movement-associated protein. This demonstrated that RLBV P4 is a functional MP. Sequence analyses revealed that P4 is a distant member of the 30K superfamily of MPs. All MPs of this family contain two highly conserved regions predicted to form β-strands, namely β1 and β2. We explored by alanine mutagenesis the role of two residues of P4 (Ile106 and Asp127) located in each of these strands. We also made the equivalent substitutions in the 29K MP of tobacco rattle virus, another member of the 30K superfamily. All substitutions abolished the ability to complement PVX movement, except for the I106A substitution in the β1 region of P4. This region has been shown to mediate membrane association of 30K MPs; our results show that it is possible to make non-conservative substitutions of a well-conserved aliphatic residue within β1 without preventing the membrane association or movement function of P4.