The cocoon into a butterfly: why the HVA22 family proteins turned out to be the reticulophagy receptors in plants?

Selective degradation of the endoplasmic reticulum (ER) by macroautophagy/autophagy (reticulophagy) is essential for maintaining ER morphology and homeostasis under environmental stresses. Several reticulophagy receptors have been identified in mammals and yeast, but their counterparts in plants have not been extensively explored yet. Recently, we demonstrated that the HVA22-family protein OsHLP1 is a reticulophagy receptor in rice plants, and its orthologs function similarly in Arabidopsis plants. In this punctum, we discuss why the HVA22 family proteins are the reticulophagy receptors in plants and how reticulophagy is highly associated with plant immune response.

OsHLP1 is an endoplasmic-reticulum-phagy receptor in rice plants.

The endoplasmic reticulum (ER) is the largest intracellular endomembrane system; it shows dynamic changes upon environmental stress. To maintain ER morphology and homeostasis under stress, the excessive ER membrane and the associated unwanted proteins can be removed via ER-phagy. Although a few ER-phagy receptors have been reported in mammals and yeast, their functional counterparts in plants remain largely unexplored. Here, we report that the HVA22 family protein OsHLP1 is an uncharacterized ER-phagy receptor in rice (Oryza sativa L.). OsHLP1 interacts with OsATG8b and recruits ER subdomains and the cargo protein OsNTL6, a negative immune regulator, to autophagosomes upon infection with the fungus Magnaporthe oryzae, which substantially activates disease resistance in rice. AtHVA22J, an Arabidopsis thaliana OsHLP1 ortholog, induced similar ER-phagy in plants. Altogether, we unraveled a conservative protein family that may act as ER-phagy receptors in higher plants, and in particular, we highlighted their roles in rice immune responses.

Nbnrp1 mediates Verticillium dahliae effector PevD1-triggered defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway in Nicotiana benthamiana.

PevD1, a fungal effector secreted by Verticillium dahliae, could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco plants. PevD1 could drastically induce the expression of Nbnrp1, which is an asparagine-rich protein (NRP) of Nicotiana benthamiana. Our previous research indicated that Nbnrp1 positively regulated PevD1-induced cell necrosis and disease resistance. In this study, we further investigated PevD1-induced immune responses in both wild-type (WT) and Nbnrp1-RNAi lines through RNA-seq, in order to reveal the underlying mechanism of Nbnrp1-modulated PevD1-induced disease resistance in N. benthamiana. Results showed that Nbnrp1-RNAi lines exhibited reduced PevD1-induced immune responses, like inhibiting H2O2 accumulation and MAPK phosphorylation. To silence Nbnrp1 inhibited the expression of PevD1-induced differential expression genes (DEGs) involved in pathways associated with sesquiterpenoid and triterpenoid biosynthesis, flavone and flavonol biosynthesis, plant-pathogen interaction and phenylpropanoid biosynthesis, etc. It is worth noting that sesquiterpene phytoalexin capsidiol accumulation were obviously decreased in Nbnrp1-RNAi plants after PevD1 treatment, accompanied with the down-expression of EAS and EAH, which were two key genes related to capsidiol biosynthesis. These results suggested that Nbnrp1 mediates PevD1-induced defense responses by regulating sesquiterpenoid phytoalexins biosynthesis pathway.

A Plant Lectin Receptor-like Kinase Phosphorylates the Bacterial Effector AvrPtoB to Dampen Its Virulence in Arabidopsis.

Plasma membrane-localized receptor-like kinases (RLKs) perceive conserved pathogen-associated molecular patterns (PAMPs) in plants, leading to PAMP-triggered immunity (PTI). The Arabidopsis thaliana lectin RLK LecRK-IX.2 has been shown to regulate the bacterial flagellin-derived peptide flg22-induced PTI. Here, we discover that Pseudomonas syringae effector AvrPtoB targets LecRK-IX.2 for degradation, which subsequently suppresses LecRK-IX.2-mediated PTI and disease resistance. However, LecRK-IX.2 can interact with and phosphorylate AvrPtoB at serine site 335 (S335). AvrPtoB self-associates in vitro and in vivo, and the association appears to be essential for its E3 ligase activity in ubiquitinating substrate in plants. Phosphorylation of S335 disrupts the self-association and as a result, phosphomimetic AvrPtoBS335D cannot ubiquitinate LecRK-IX.2 efficiently, leading to the compromised virulence of AvrPtoB in suppressing PTI responses. flg22 enhances AvrPtoB S335 phosphorylation by inducing the expression and activating of LecRK-IX.2. Our study demonstrates that host RLKs can modify pathogen effectors to dampen their virulence and undermine their ability in suppressing PTI.

Tyrosine phosphorylation of the lectin receptor-like kinase LORE regulates plant immunity.

Plant pattern recognition receptors (PRRs) perceive pathogen-associated molecular patterns (PAMPs) to activate immune responses. Medium-chain 3-hydroxy fatty acids (mc-3-OH-FAs), which are widely present in Gram-negative bacteria, were recently shown to be novel PAMPs in Arabidopsis thaliana. The Arabidopsis PRR LIPOOLIGOSACCHARIDE-SPECIFIC REDUCED ELICITATION (LORE) is a G-type lectin receptor-like kinase that recognizes mc-3-OH-FAs and subsequently mounts an immune response; however, the mechanisms underlying LORE activation and downstream signaling are unexplored. Here, we report that one of the mc-3-OH-FAs, 3-OH-C10:0, induces phosphorylation of LORE at tyrosine residue 600 (Y600). Phosphorylated LORE subsequently trans-phosphorylates the receptor-like cytoplasmic kinase PBL34 and its close paralogs, PBL35 and PBL36, and therefore activates plant immunity. Phosphorylation of LORE Y600 is required for downstream phosphorylation of PBL34, PBL35, and PBL36. However, the Pseudomonas syringae effector HopAO1 targets LORE, dephosphorylating the tyrosine-phosphorylated Y600 and therefore suppressing the immune response. These observations uncover the mechanism by which LORE mediates signaling in response to 3-OH-C10:0 in Arabidopsis.

Verticillium dahliae PevD1, an Alt a 1-like protein, targets cotton PR5-like protein and promotes fungal infection.

Alt a 1 family proteins (AA1s) have only been observed in the Dothideomycetes and Sordariomycetes classes of fungi, and their biological functions have remained poorly understood. Verticillium dahliae, a soil-borne pathogen that causes plant wilt disease, secretes hundreds of proteins during the process of pathogenic infection, including the AA1 member PevD1. In this study, we found that the pevd1 transcript was present in all of the hosts studied (cotton, Arabidopsis, tomato, and tobacco) and showed elevated expression throughout the infection process. Furthermore, pevd1 knockout mutants displayed attenuated pathogenicity compared with the wild-type (WT) strain and complemented strains in hosts. A partner protein of PevD1, pathogenesis-related protein 5 (PR5)-like protein GhPR5, was isolated from cotton (Gossypium hirsutum) plants by co-purification assays, and the PevD1-GhPR5 interaction was determined to be localized in the C-terminus (PevD1b, amino acids residues 113-155) by pull-down and yeast two-hybrid techniques. Re-introduction of the pevd1b gene into a pevd1 knockout mutant resulted in restoration of the virulence phenotype to WT levels. In addition, PevD1b, which is similar to PevD1, decreased the antifungal activity of GhPR5 in vitro. Our findings reveal an infection strategy in which V. dahliae secretes PevD1 to inhibit GhPR5 antifungal activity in order to overcome the host defence system.

Lignin metabolism involves Botrytis cinerea BcGs1- induced defense response in tomato.

BACKGROUND: BcGs1, a cell wall-degrading enzyme (CWDE), was originally derived from Botrytis cinerea. Our previous study revealed that BcGs1 could trigger defense responses and protect plants against various pathogens. We researched the defense response mechanism underlying this BcGs1 elicitation in tomato. RESULTS: We revealed that the two domains were required for BcGs1's full necrosis activity. According to analysis and quantitative real-time PCR of the up-regulated proteins and genes filtered by iTRAQ-based quantitative proteome approach, oxidative metabolism and phenylpropanoid metabolism were speculated to be involved in BcGs1-triggered defense response in tomato. Furthermore, experimental evidence showed that BcGs1 triggered reactive oxygen species (ROS) burst and increased the level of phenylalanine-ammonia lyase (PAL) and peroxidase (POD) enzyme activity, as well as lignin accumulation. Moreover, histochemical analysis revealed that infiltration of BcGs1 in tomato leaves exhibited cell wall thickening compared with untreated plants. CONCLUSIONS: The results suggested that BcGs1 activated the basal defense response included lignin metabolism contributed to BcGs1-induced resistance to Botrytis. cinerea infection in tomato.

An Asparagine-Rich Protein Nbnrp1 Modulate Verticillium dahliae Protein PevD1-Induced Cell Death and Disease Resistance in Nicotiana benthamiana.

PevD1 is a fungal protein secreted by Verticillium dahliae. Our previous researches showed that this protein could induce hypersensitive responses-like necrosis and systemic acquired resistance (SAR) in cotton and tobacco. To understand immune activation mechanisms whereby PevD1 elicits defense response, the yeast two-hybrid (Y2H) assay was performed to explore interacting protein of PevD1 in Arabidopsis thaliana, and a partner AtNRP (At5g42050) was identified. Here, AtNRP homolog in Nicotiana benthamiana was identified and designated as Nbnrp1. The Nbnrp1 could interact with PevD1 via Y2H and bimolecular fluorescence complementation (BiFC) analyses. Moreover, truncated protein binding assays demonstrated that the C-terminal 132 amino acid (development and cell death, DCD domain) of Nbnrp1 is required for PevD1-Nbnrp1 interaction. To further investigate the roles of Nbnrp1 in PevD1-induced defense response, Nbnrp1-overexpressing and Nbnrp1-silence transgenic plants were generated. The overexpression of Nbnrp1 conferred enhancement of PevD1-induced necrosis activity and disease resistance against tobacco mosaic virus (TMV), bacterial pathogen Pseudomonas syringae pv. tabaci and fungal pathogen V. dahliae. By contrast, Nbnrp1-silence lines displayed attenuated defense response compared with the wild-type. It is the first report that an asparagine-rich protein Nbnrp1 positively regulated V. dahliae secretory protein PevD1-induced cell death response and disease resistance in N. benthamiana.

The Verticillium dahliae SnodProt1-Like Protein VdCP1 Contributes to Virulence and Triggers the Plant Immune System.

During pathogenic infection, hundreds of proteins that play vital roles in the Verticillium dahliae-host interaction are secreted. In this study, an integrated proteomic analysis of secreted V. dahliae proteins was performed, and a conserved secretory protein, designated VdCP1, was identified as a member of the SnodProt1 phytotoxin family. An expression analysis of the vdcp1 gene revealed that the transcript is present in every condition studied and displays elevated expression throughout the infection process. To investigate the natural role of VdCP1 in V. dahliae, two vdcp1 knockout mutants and their complementation strains were generated. Bioassays of these mutants revealed no obvious phenotypic differences from the wild-type (WT) in terms of mycelial growth, conidial production or mycelial/spore morphology. However, compared with the WT, the vdcp1 knockout mutants displayed attenuated pathogenicity in cotton plants. Furthermore, treating plants with purified recombinant VdCP1 protein expressed in Pichia pastoris induced the accumulation of reactive oxygen species (ROS), expression of several defense-related genes, leakage of ion electrolytes, enhancement of defense-related enzyme activity and production of salicylic acid. Moreover, VdCP1 conferred resistance to Botrytis cinerea and Pseudomonas syringae pv. tabaci in tobacco and to V. dahliae in cotton. Further research revealed that VdCP1 possesses chitin-binding properties and that the growth of vdcp1 knockout mutants was more affected by treatments with chitinase, indicating that VdCP1 could protect V. dahliae cell wall from enzymatic degradation, which suggests an effector role of VdCP1 in infecting hosts.

The Magnaporthe oryzae Alt A 1-like protein MoHrip1 binds to the plant plasma membrane.

MoHrip1, a protein isolated from Magnaporthe oryzae, belongs to the Alt A 1 (AA1) family. mohrip1 mRNA levels showed inducible expression throughout the infection process in rice. To determine the location of MoHrip1 in M. oryzae, a mohrip1-gfp mutant was generated. Fluorescence microscopy observations and western blotting analysis showed that MoHrip1 was both present in the secretome and abundant in the fungal cell wall. To obtain MoHrip1 protein, we carried out high-yield expression of MoHrip1 in Pichia pastoris. Treatment of tobacco plants with MoHrip1 induced the formation of necrosis, accumulation of reactive oxygen species and expression of several defense-related genes, as well as conferred disease resistance. By fusion to green fluorescent protein, we showed that MoHrip1 was able to bind to the tobacco and rice plant plasma membrane, causing rapid morphological changes at the cellular level, such as cell shrinkage and chloroplast disorganization. These findings indicate that MoHrip1 is a microbe-associated molecular pattern that is perceived by the plant immune system. This is the first study on an AA1 family protein that can bind to the plant plasma membrane.

Comparison of cerato-platanin family protein BcSpl1 produced in Pichia pastoris and Escherichia coli.

The Botrytis cinerea BcSpl1 protein is a member of the cerato-platanin family, and consists of 137 amino acids and two disulfide bridges. This protein induces the onset of necrosis in infiltrated plant hosts. Recombinant BcSpl1 proteins produced in Pichia pastoris (pBcSpl1) and Escherichia coli (eBcSpl1) were initially compared regarding their abilities to induce necrosis and systemic acquired resistance (SAR). The pBcSpl1 and eBcSpl1 treatments led to the development of necrotic lesions on tomato leaves, and provided tomato plants with SAR to B. cinerea. The lesion area of leaves infiltrated with the BcSpl1 proteins decreased by 22.7% (pBcSpl1) and 21.8% (eBcSpl1). Additionally, eBcSpl1 up-regulated the expression levels of some defense-related genes, including PR-1a, prosystemin, PI I, and PI II, as well as SIPK and TPK1b, which encode two protein kinases. Furthermore, eBcSpl1 exhibited chitin-binding properties. Our data revealed that the E. coli expression system produces higher BcSpl1 yields than the P. pastoris system. This high-yield expression of BcSpl1 may be relevant for future large-scale applications of this elicitor to improve crop production.