Activation of MAPK-mediated immunity by phosphatidic acid in response to positive-strand RNA viruses.

Increasing evidence suggests that mitogen-activated protein kinase (MAPK) cascades play a crucial role in plant defense against viruses. However, the mechanisms that underlie the activation of MAPK cascades in response to viral infection remain unclear. In this study, we discovered that phosphatidic acid (PA) represents a major class of lipids that respond to Potato virus Y (PVY) at an early stage of infection. We identified NbPLDα1 (Nicotiana benthamiana phospholipase Dα1) as the key enzyme responsible for increased PA levels during PVY infection and found that it plays an antiviral role. 6K2 of PVY interacts with NbPLDα1, leading to elevated PA levels. In addition, NbPLDα1 and PA are recruited by 6K2 to membrane-bound viral replication complexes. On the other hand, 6K2 also induces activation of the MAPK pathway, dependent on its interaction with NbPLDα1 and the derived PA. PA binds to WIPK/SIPK/NTF4, prompting their phosphorylation of WRKY8. Notably, spraying with exogenous PA is sufficient to activate the MAPK pathway. Knockdown of the MEK2-WIPK/SIPK-WRKY8 cascade resulted in enhanced accumulation of PVY genomic RNA. 6K2 of Turnip mosaic virus and p33 of Tomato bushy stunt virus also interacted with NbPLDα1 and induced the activation of MAPK-mediated immunity. Loss of function of NbPLDα1 inhibited virus-induced activation of MAPK cascades and promoted viral RNA accumulation. Thus, activation of MAPK-mediated immunity by NbPLDα1-derived PA is a common strategy employed by hosts to counteract positive-strand RNA virus infection.

The Great Game between Plants and Viruses: A Focus on Protein Homeostasis.

Plant viruses are tiny pathogenic obligate parasites that cause significant damage to global crop production. They exploit and manipulate the cellular components of host plants to ensure their own survival. In response, plants activate multiple defense signaling pathways, such as gene silencing and plant hormone signaling, to hinder virus propagation. Growing evidence suggests that the regulation of protein homeostasis plays a vital role in the ongoing battle between plants and viruses. The ubiquitin-proteasome-degradation system (UPS) and autophagy, as two major protein-degradation pathways, are widely utilized by plants and viruses in their arms race. One the one hand, these pathways act as essential components of plant's antiviral defense system by facilitating the degradation of viral proteins; on the other hand, viruses exploit the UPS and autophagy to create a favorable intracellular environment for viral infection. This review aims to provide a comprehensive summary of the events involved in protein homeostasis regulation during viral infection in plants. Gaining knowledge in this area will enhance our understanding of the complex interplay between plants and viruses.

Cucurbit Chlorotic Yellows Virus p22 Protein Interacts with Cucumber SKP1LB1 and Its F-Box-Like Motif Is Crucial for Silencing Suppressor Activity.

Plants use RNA silencing as a defense against viruses. In response, viruses encode various RNA silencing suppressors to counteract the antiviral silencing. Here, we identified p22 as a silencing suppressor of cucurbit chlorotic yellows crinivirus and showed that p22 interacts with CsSKP1LB1, a Cucumis sativus ortholog of S-phase kinase-associated protein 1 (SKP1). The F-box-like motif of p22 was identified through sequence analysis and found to be necessary for the interaction using a yeast two-hybrid assay. The involvement of the F-box-like motif in p22 silencing suppressor activity was determined. Proteomics analysis of Nicotiana benthamiana leaves expressing p22, and its F-box-like motif deletion mutant showed 228 differentially expressed proteins and five enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways: ABC transporters, sesquiterpenoid and triterpenoid biosynthesis, ubiquitin-mediated proteolysis, riboflavin metabolism, and cysteine and methionine metabolism. Collectively, our results demonstrate the interaction between p22 and CsSKP1LB1 and show that the deletion of F-box-like motif inhibits p22 silencing suppressor activity. The possible pathways regulated by the p22 through the F-box-like motif were identified using proteomics analysis.

Purification and Characterization of a Secretory Alkaline Metalloprotease with Highly Potent Antiviral Activity from Serratia marcescens Strain S3.

In this study we report a secretory protein that was purified from Serratia marcescens strain S3 isolated from soil from the tobacco rhizosphere. Subsequent mass spectrometry and annotation characterized the protein as secretory alkaline metalloprotease (SAMP). SAMP plays a crucial role in inhibiting Tobacco mosaic virus (TMV). Transmission electron microscopy (TEM), dynamic light scattering (DLS), confocal microscopy, and microscale thermophoresis (MST) were employed to investigate the anti-TMV mechanism of SAMP. Our results demonstrated that SAMP, as a hydrolytic metal protease, combined and hydrolyzed TMV coat proteins to destroy the virus particles. This study is the first to investigate the antiviral effects of a S. marcescens metalloprotease, and our finding suggests that S. marcescens-S3 may be agronomically useful as a disease-controlling factor active against Tobacco mosaic virus.

Complete genome sequence of an alternavirus from the phytopathogenic fungus Fusarium incarnatum.

A novel double-stranded RNA (dsRNA) mycovirus, named "Fusarium incarnatum alternavirus 1" (FiAV1), was found in Fusarium incarnatum strain LY003-07, the causal agent of peony root rot. The complete genome of FiAV1 is composed of three dsRNA segments, dsRNA1 (3548 nt), dsRNA2 (2514 nt), and dsRNA3 (2498 nt), with one open reading frame on each of their positive-sense strands. As found in other viruses of the proposed family Alternaviridae, the positive-sense strand of each genomic dsRNA of FiAV1 has a poly(A) tail and the 5'-terminal nonamer sequence 5'-GGCTGTGTG-3'. Based on a multiple sequence alignment and phylogenetic analysis based on the RdRps amino acid sequence, FiAV1 is suggested to be a new strain of a potential new species, for which the name "Fusarium alternavirus 1" is proposed, that includes Fusarium poae alternavirus 1 (FpAV1) and Fusarium graminearum alternavirus 1 (FgAV1/AH11) of the proposed family "Alternaviridae". This is the first report of a mycovirus of the proposed family "Alternaviridae" that infects F. incarnatum.

CLC-Nt1 affects Potato Virus Y infection via regulation of endoplasmic reticulum luminal Ph.

Chloride channel (CLC) proteins are important anion transporters conserved in organisms ranging from bacteria and yeast to plants and animals. According to sequence comparison, some plant CLCs are predicted to function as Cl- /H+ antiporters, but not Cl- channels. However, no direct evidence was provided to verify the role of these plant CLCs in regulating the pH of the intracellular compartment. We identified tobacco CLC-Nt1 interacting with the Potato virus Y (PVY) 6K2 protein. To investigate its physiological function, homologous genes of CLC-Nt1 in Nicotiana benthamiana were knocked out using the CRISPR/Cas9 system. Complementation experiments were subsequently performed by expression of wild-type or point-mutated CLC-Nt1 in knockout mutants. The data presented herein demonstrate that CLC-Nt1 is localized at endoplasmic reticulum (ER). Using a pH-sensitive fluorescent protein (pHluorin), we found that loss of CLC-Nt1 function resulted in a decreased ER luminal pH. Secreted GFP (secGFP) was retained mostly in ER in knockout mutants, indicating that CLC-Nt1 is also involved in protein secretion. PVY infection induced a rise in ER luminal pH, which was dependent on functional CLC-Nt1. By contrast, loss of CLC-Nt1 function inhibited PVY intracellular replication and systemic infection. We propose that PVY alters ER luminal pH for infection in a CLC-Nt1-dependent manner.

First comprehensive proteome analysis of lysine crotonylation in seedling leaves of Nicotiana tabacum.

Histone crotonylation is a new lysine acylation type of post-translational modification (PTM) enriched at active gene promoters and potential enhancers in yeast and mammalian cells. However, lysine crotonylation in nonhistone proteins and plant cells has not yet been studied. In the present study, we performed a global crotonylation proteome analysis of Nicotiana tabacum (tobacco) using high-resolution LC-MS/MS coupled with highly sensitive immune-affinity purification. A total of 2044 lysine modification sites distributed on 637 proteins were identified, representing the most abundant lysine acylation proteome reported in the plant kingdom. Similar to lysine acetylation and succinylation in plants, lysine crotonylation was related to multiple metabolism pathways, such as carbon metabolism, the citrate cycle, glycolysis, and the biosynthesis of amino acids. Importantly, 72 proteins participated in multiple processes of photosynthesis, and most of the enzymes involved in chlorophyll synthesis were modified through crotonylation. Numerous crotonylated proteins were implicated in the biosynthesis, folding, and degradation of proteins through the ubiquitin-proteasome system. Several crotonylated proteins related to chromatin organization are also discussed here. These data represent the first report of a global crotonylation proteome and provide a promising starting point for further functional research of crotonylation in nonhistone proteins.