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.

First Report of Potato Viruses Infecting Lamium purpureum in Ukraine

Weeds as reservoirs for destructive plant pathogens have a significant impact on the viral epidemiology, ecology and, as a result, on local economy, and are therefore being investigated in many parts of the world. Thus, the aim of this study was to investigate virus occurrence in red dead-nettle plants (Lamium purpureum L.) widespread in urban and field conditions throughout the in the Kyiv region of Ukraine. Methods. Field crop observations, visual diagnosis, biological testing of the virus, immunoassay (ELISA), polymerase chain reaction with reverse transcription (RT-PCR), sanger sequencing of partial genome sequences of PVX, PVY, PVS, PVM. Results. The results obtained in the study indicate that Lamium plants could be alternative weed hosts of number important viral diseases including potatoes and other vegetables. Serological and molecular test results evidence plants were infected by Potato virus X, Potato virus Y, Potato virus M, Potato virus S and therefore Lamium L. species can serve as a potential source of inoculum for wide range of vegetables and ornamentals. This study is the first report of Lamium plants being naturally infected with Potato virus M and Potato virus S in central Europe. Conclusions. These plants are alternative host of mixed infection with viruses belonging to different families: Alphaflexiviridae, Betaflexiviridae and Potyviridae.

Pre-Symptomatic Detection of Viral Infection in Tobacco Leaves Using PAM Fluorometry

Chlorophyll fluorescence imaging was used to study potato virus X (PVX) infection of Nicotiana benthamiana. Infection-induced changes in chlorophyll fluorescence parameters (quantum yield of photosystem II photochemistry (ΦPSII) and non-photochemical fluorescence quenching (NPQ)) in the non-inoculated leaf were recorded and compared with the spatial distribution of the virus detected by the fluorescence of GFP associated with the virus. We determined infection-related changes at different points of the light-induced chlorophyll fluorescence kinetics and at different days after inoculation. A slight change in the light-adapted steady-state values of ΦPSII and NPQ was observed in the infected area of the non-inoculated leaf. In contrast to the steady-state parameters, the dynamics of ΦPSII and NPQ caused by the dark–light transition in healthy and infected areas differed significantly starting from the second day after the detection of the virus in a non-inoculated leaf. The coefficients of correlation between chlorophyll fluorescence parameters and virus localization were 0.67 for ΦPSII and 0.76 for NPQ. In general, the results demonstrate the possibility of reliable pre-symptomatic detection of the spread of a viral infection using chlorophyll fluorescence imaging.

Turnip mosaic virus P1 suppresses JA biosynthesis by degrading cpSRP54 that delivers AOCs onto the thylakoid membrane to facilitate viral infection

Jasmonic acid (JA) is a crucial hormone in plant antiviral immunity. Increasing evidence shows that viruses counter this host immune response by interfering with JA biosynthesis and signaling. However, the mechanism by which viruses affect JA biosynthesis is still largely unexplored. Here, we show that a highly conserved chloroplast protein cpSRP54 was downregulated in Nicotiana benthamiana infected by turnip mosaic virus (TuMV). Its silencing facilitated TuMV infection. Furthermore, cpSRP54 interacted with allene oxide cyclases (AOCs), key JA biosynthesis enzymes, and was responsible for delivering AOCs onto the thylakoid membrane (TM). Interestingly, TuMV P1 protein interacted with cpSRP54 and mediated its degradation via the 26S proteosome and autophagy pathways. The results suggest that TuMV has evolved a strategy, through the inhibition of cpSRP54 and its delivery of AOCs to the TM, to suppress JA biosynthesis and enhance viral infection. Interaction between cpSRP54 and AOCs was shown to be conserved in Arabidopsis and rice, while cpSRP54 also interacted with, and was degraded by, pepper mild mosaic virus (PMMoV) 126 kDa protein and potato virus X (PVX) p25 protein, indicating that suppression of cpSRP54 may be a common mechanism used by viruses to counter the antiviral JA pathway.

Pontin/Reptin-associated complexes differentially impact plant development and viral pathology

Pontin and Reptin are essential eukaryotic AAA+ ATPases that work together in several multiprotein complexes, contributing to chromatin remodeling and TARGET OF RAPAMCYIN (TOR) kinase complex assembly, among other functions. Null alleles of pontin or reptin are gametophyte lethal in plants, which has hindered studies of their crucial roles in plant biology. Here, we used virus-induced gene silencing (VIGS) to interrogate the functions of Pontin and Reptin in plant growth and physiology, focusing on Nicotiana benthamiana, a model species for the agriculturally significant Solanaceae family. Silencing either Pontin or Reptin caused pleiotropic developmental and physiological reprogramming, including aberrant leaf shape, reduced apical growth, delayed flowering, increased branching, chlorosis, and decreased spread of the RNA viruses Tobacco mosaic virus (TMV) and Potato virus X (PVX). To dissect these pleiotropic phenotypes, we took a comparative approach and silenced expression of key genes that encode subunits of each of the major Pontin/Reptin-associated chromatin remodeling or TOR complexes (INO80, SWR-C/PIE1, TIP60, TOR, and TELO2). We found that many of the pontin/reptin phenotypes could be attributed specifically to disruption of one of these complexes, with tip60 and tor knockdown plants each phenocopying a large subset of pontin/reptin phenotypes. We conclude that Pontin/Reptin complexes are crucial for proper plant development, physiology, and stress responses, highlighting the multifaceted roles these conserved enzymes have evolved in eukaryotic cells.

Robust Agrobacterium-Mediated Transient Expression in Two Duckweed Species (Lemnaceae) Directed by Non-replicating, Replicating, and Cell-to-Cell Spreading Vectors

Plant-based transient expression systems have recognized potential for use as rapid and cost-effective alternatives to expression systems based on bacteria, yeast, insect, or mammalian cells. The free-floating aquatic plants of the Lemnaceae family (duckweed) have compact architecture and can be vegetatively propagated on low-cost nutrient solutions in aseptic conditions. These features provide an economically feasible opportunity for duckweed-based production of high-value products via transient expression of recombinant products in fully contained, controlled, aseptic and bio-safe conditions in accordance with the requirements for pharmaceutical manufacturing and environmental biosafety. Here, we demonstrated Agrobacterium-mediated high-yield transient expression of a reporter green fluorescent protein using deconstructed vectors based on potato virus X and sweet potato leaf curl virus, as well as conventional binary vectors, in two representatives of the Lemnaceae (Spirodela polyrhiza and Landoltia punctata). Aseptically cultivated duckweed populations yielded reporter protein accumulation of >1 mg/g fresh biomass, when the protein was expressed from a deconstructed potato virus X-based vector, which is capable of replication and cell-to-cell movement of the replicons in duckweed. The expression efficiency demonstrated here places duckweed among the most efficient host organisms for plant-based transient expression systems, with the additional benefits of easy scale-up and full containment.

In virto and in vivo Phosphorylation of a Coat Protein of Potato Virus X

At the present stage of development of plant virology the study of molecular mechanisms of regulation, translation and replication of viral RNA is of great interest. Potato virus X (PVX) RNA in viral particles is not available for in vitro translation, but acquires the ability to be translated as a result of shell protein phosphorylation. The aim of our study was to investigate the conditions of phosphorylation of the PVX coat protein in in vitro and in vivo systems, as well as the effect of EDTA and CaCl2 on the phosphorylation in vitro. Methods. The PVX coat protein was obtained by the guanidine chloride method. The kinase activity of PVX protein in vitro was determined in a standard reaction mixture containing Mn2+ ions, 0.8 mM EDTA, and 2 micro Ci 32P ATP (3000 Ci/mM). Phosphorylation of the protein in vivo was carried out by immersing Datura stramonium leaves with symptoms of PVX infection in water containing К3PO4 32P. After isolation of PVX from the leaves, the viral coat protein was fractionated by SDS-PAAG electrophoresis. Fractions of the protein were transferred from the gel by contact manner on a nitrocellulose filter. The PVX coat protein was detected by immunoblotting using immunoglobulins to PVX coat protein and rabbit antibodies labeled with peroxidase. The inclusion of labeled phosphorus in the PVX protein was detected by radioautography. Results. The PVX coat protein was phosphorylated in vitro in a standard incubation medium containing (gamma -32P) ATP. In contrast, the PVX coat protein cannot be phosphorylated in the same conditions in the presence of (alpha-32P) ATP. In vivo phosphorylated PVX coat protein was detected by exposing nitrocellulose filter with immunoblot on X-ray film. Additionally, it was found that the presence of 10 mm EDTA and 10 mm CaCl2 inhibited the process of the PVX coat protein phosphorylation in vitro. Conclusions. The coat protein of potato virus X is able to phosphorylate in vitro and in vivo systems. The terminal ATP phosphate plays a major role in the phosphorylation of the PVX coat protein. The presence of EDTA and Ca2+ influences on the process of protein phosphorylation in vitro. These agents are able to inhibit the process of phosphorylation of the PVX coat protein. Thus, the phenomenon of phosphorylation of the PVX coat protein apparently indicates about its participation in the regulation of the virus reproduction in the infected cell.