Background: Major latex proteins (MLPs) belong to the MLP subfamily in Bet v 1 protein family and respond to both biotic and abiotic stresses, which play critical roles in plant disease resistance. As the type species of widely distributed and economically devastating Potyvirus, Potato virus Y (PVY) is one of the major constraints to important crop plants including tobacco (Nicotiana benthamiana) worldwide. Despite the great losses owing to PVY infection in tobacco, there is no previous study investigating the potential role of MLPs in developing resistance to viral infection. Results: In this study, for the first time we have identified and functionally analyzed the MLP-like protein 28 from N. benthamiana, denoted as NbMLP28 and investigated its role in conferring resistance to N. benthamiana against PVY infection. NbMLP28 was localized to the plasmalemma and nucleus, with the highest level in the root. NbMLP28 gene was hypothesized to be triggered by PVY infection and was highly expressed in jasmonic acid (JA) signaling pathway. Further validation was achieved through silencing of NbMLP28 through virus-induced gene silencing (VIGS) that rendered N. benthamiana plants more vulnerable to PVY infection, contrary to overexpression that enhanced resistance. Conclusions: Taken together, this is the first study describing the role of NbMLP28 in tobacco against PVY infection and provide a pivotal point towards obtaining pathogen-resistant tobacco varieties through constructing new candidate genes of MLP subfamily.

The ubiquitin–proteasome system (UPS) plays an important role in virus–host interactions. However, the mechanism by which the UPS is involved in innate immunity remains unclear. In this study, we identified a novel major latex protein-like protein 43 (NbMLP43) that conferred resistance to Nicotiana benthamiana against potato virus Y (PVY) infection. PVY infection strongly induced NbMLP43 transcription but decreased NbMLP43 at the protein level. We verified that B-box zinc finger protein 24 (NbBBX24) interacted directly with NbMLP43 and that NbBBX24, a light responsive factor, acted as an essential intermediate component targeting NbMLP43 for its ubiquitination and degradation via the UPS. PVY, tobacco mosaic virus, (TMV) and cucumber mosaic virus (CMV) infections could promote NbMLP43 ubiquitination and proteasomal degradation to enhance viral infection. Ubiquitination occurred at lysine 38 (K38) within NbMLP43, and non-ubiquitinated NbMLP43(K38R) conferred stronger resistance to RNA viruses. Overall, our results indicate that the novel NbMLP43 protein is a target of the UPS in the competition between defense and viral anti-defense and enriches existing theoretical studies on the use of UPS by viruses to promote infection.

Lysine acetylation (K-ac), a reversible PTM, plays an essential role in various biological processes, including those involving metabolic pathways, pathogen resistance, and transcription, in both prokaryotes and eukaryotes. TMV, the major factor that causes the poor quality of Solanaceae crops worldwide, directly alters many metabolic processes in tobacco. However, the extent and function of K-ac during TMV infection have not been determined. The validation test to detect K-ac level and viral expression after TMV infection and Nicotinamide (NAM) treatment clarified that acetylation was involved in TMV infection. Furthermore, we comprehensively analyzed the changes in the proteome and acetylome of TMV-infected tobacco (Nicotiana benthamiana) seedlings via LC-MS/MS in conjunction with highly sensitive immune-af inity purification. In total, 2082 lysine-acetylated sites on 1319 proteins differentially expressed in response to TMV infection were identified. Extensive bioinformatic studies disclosed changes in acetylation of proteins engaged in cellular metabolism and biological processes. The vital influence of Kac in fatty acid degradation and alpha-linolenic acid metabolism was also revealed in TMV-infected seedlings. This study first revealed Kac information in N. benthamiana under TMV infection and expanded upon the existing landscape of acetylation in pathogen infection.

BZIP transcription factors (TFs) play an important role in plant development and stress. This study identified 79 bZIP TFs in Nicotiana benthamiana , which were divided into ten subgroups, i.e., A, B, C, D, E, F, G, H, I, and S. bZIP60, whose promoter sequence contains cis -acting elements involved in stress response and pathogen infection, belonged to group H. The IRE1/bZIP60 pathway of the unfolded protein response (UPR) signal, a cellular defense signal, plays dual roles upon viral infection. This study confirmed that potato virus Y (PVY) infection induced endoplasmic reticulum (ER) stress-related UPR by changing ER morphology and upregulating UPR genes, (such as BLP4 , bZIP60 , bZIP28 , and NAC089 , referring to Li et al . 2017). Furthermore, bZIP60 was upregulated by spraying of salicylic acid and jasmonic acid, which are essential for systemic resistance against plant viruses. bZIP60 silencing resulted in delayed growth of N. benthamiana , increased PVY accumulation, and impaired UPR gene expression upon viral infection. bZIP60 overexpression decreased PVY infection and replication. Therefore, upon PVY infection, bZIP60 is a positive regulator that promotes plant growth and induces cellular defense signals of UPR.