Protein stability governed by α1-2 helices in Pvr4 is essential for localization and cell death.

Plant nucleotide-binding domain leucine-rich-repeat receptor (NLR) confers disease resistance to various pathogens by recognizing effectors derived from the pathogen. Previous studies have shown that overexpression of the CC domain in several NLRs triggers cell death, implying that the CC domain plays an important role as a signaling module. However, how CC domain transduces immune signals remains largely unknown. A Potyvirus-resistant NLR protein, Pvr4, possesses a CC domain (CCPvr4 ) that induces cell death upon transient overexpression in Nicotiana benthamiana. In this study, loss-of-function mutants were generated by error-prone PCR-based random mutagenesis to understand the molecular mechanisms underlying CCPvr4 -mediated cell death. Cell biology and biochemical studies revealed that M16 and Q52 in the α1 and α2 helices, respectively, are crucial for protein stability, and mutation of these residues disrupts localization to the plasma membrane and oligomerization activity. The increase of the protein stability of these mutants by tagging a green fluorescent protein (GFP) variant led to restoration of cell death-inducing activity and plasma membrane localization. Another mutant, I7E in the very N-terminal region, lost cell death-inducing activity by weakening the interaction with plasma membrane H+ -ATPase compared to CCPvr4 , although the protein remained in the plasma membrane. Moreover, most of the mutated residues are on the outer surface of the funnel shape in the predicted pentameric CCPvr4 , implying that the disordered N-terminal region plays a crucial role in association with PMA as well as targeting to the plasma membrane. This work could provide insights into the molecular mechanisms of cell death induced by NLR immune receptors.

In-silico evolutionary analysis of plant-OBERON proteins during compatible MYMV infection in respect of improving host resistance.

Yellow mosaic disease (YMD) of pulses caused by mungbean yellow mosaic virus is a major threat to crop production. An infection that is compatible with regulating and interacting host proteins and the virus causes YMD. Oberon families of proteins OBE1-4 and VIN1-4 are imperative for plants, functions in meristem and vascular development, and were also regulated during compatible disease infection. Furthermore, in-silico expression results suggested the involvement of OBE1 and OBE2 proteins during virus infection of Vigna, Arabidopsis and soybean. Moreover, a common ancestor for the meristem and virus movement related Oberons was inferred through phylogenetic analysis. Protein interaction studies showed three amino acids (Aspartate, glutamate and lysine) in the plant homeodomain (PHD), involved in interaction with the N-terminal region of the virus movement protein and were also conserved in both monocot and dicots. Additionally, major differences in the nuclear localization signal (NLS) showing clade specific conservation and significant variation between dicots and monocots were ascertained in meristem and virus movement related Oberons. Consequently, a combination of PHD, CCD and their interactions with the VPg viral domain increases the susceptibility to YMD. Further, modification in the NLS regions of the viral movement clade Oberons, to knock out allele generation in the OBE1 and OBE2 homologs through genome-editing approaches could be established as alternate strategies for the improvement of host resistance and control yellow mosaic disease in plants, especially in pulse crops.

Structural basis of NLR activation and innate immune signalling in plants.

Animals and plants have NLRs (nucleotide-binding leucine-rich repeat receptors) that recognize the presence of pathogens and initiate innate immune responses. In plants, there are three types of NLRs distinguished by their N-terminal domain: the CC (coiled-coil) domain NLRs, the TIR (Toll/interleukin-1 receptor) domain NLRs and the RPW8 (resistance to powdery mildew 8)-like coiled-coil domain NLRs. CC-NLRs (CNLs) and TIR-NLRs (TNLs) generally act as sensors of effectors secreted by pathogens, while RPW8-NLRs (RNLs) signal downstream of many sensor NLRs and are called helper NLRs. Recent studies have revealed three dimensional structures of a CNL (ZAR1) including its inactive, intermediate and active oligomeric state, as well as TNLs (RPP1 and ROQ1) in their active oligomeric states. Furthermore, accumulating evidence suggests that members of the family of lipase-like EDS1 (enhanced disease susceptibility 1) proteins, which are uniquely found in seed plants, play a key role in providing a link between sensor NLRs and helper NLRs during innate immune responses. Here, we summarize the implications of the plant NLR structures that provide insights into distinct mechanisms of action by the different sensor NLRs and discuss plant NLR-mediated innate immune signalling pathways involving the EDS1 family proteins and RNLs.

Pm21 CC domain activity modulated by intramolecular interactions is implicated in cell death and disease resistance.

Nucleotide-binding (NB) leucine-rich repeat (LRR) receptors (NLRs) provide resistance against several plant pathogens. We previously cloned the wheat powdery mildew resistance gene Pm21, which encodes a coiled-coil (CC) NLR that confers broad-spectrum resistance against Blumeria graminis f. sp. tritici. Here, we report comprehensive biochemical and functional analyses of Pm21 CC domain in Nicotiana benthamiana. Transient overexpression assay suggested that only the extended CC (eCC, amino acid residues 1-159) domain has cell-death-inducing activity, whereas the CC-containing truncations, including CC-NB and CC-NB-LRR, do not induce cell-death responses. Coimmunoprecipitation (Co-IP) assay showed that the eCC domain self-associates and interacts with the NB and LRR domains in planta. These results imply that the activity of the eCC domain is inhibited by the intramolecular interactions of different domains in the absence of pathogens. We found that the LRR domain plays a crucial role in D491V-mediated full-length (FL) Pm21 autoactivation. Some mutations in the CC domain leading to the loss of Pm21 resistance to powdery mildew impaired the CC activity of cell-death induction. Two mutations (R73Q and E80K) interfered with D491V-mediated Pm21 autoactivation without affecting the cell-death-inducing activity of the eCC domain. Notably, some susceptible mutants harbouring mutations in the CC domain still exhibited cell-death-inducing activity. Taken together, these results implicate the CC domain of Pm21 in cell-death signalling and disease-resistance signalling, which are potentially independent of each other.

Binding of Avibirnavirus VP3 to the PIK3C3-PDPK1 complex inhibits autophagy by activating the AKT-MTOR pathway.

Macroautophagy/autophagy is a host natural defense response. Viruses have developed various strategies to subvert autophagy during their life cycle. Recently, we revealed that autophagy was activated by binding of Avibirnavirus to cells. In the present study, we report the inhibition of autophagy initiated by PIK3C3/VPS34 via the PDPK1-dependent AKT-MTOR pathway. Autophagy detection revealed that viral protein VP3 triggered inhibition of autophagy at the early stage of Avibirnavirus replication. Subsequent interaction analysis showed that the CC1 domain of VP3 disassociated PIK3C3-BECN1 complex by direct interaction with BECN1 and blocked autophagosome formation, while the CC3 domain of VP3 disrupted PIK3C3-PDPK1 complex via directly binding to PIK3C3 and inhibited both formation and maturation of autophagosome. Furthermore, we found that PDPK1 activated AKT-MTOR pathway for suppressing autophagy via binding to AKT. Finally, we proved that CC3 domain was critical for role of VP3 in regulating replication of Avibirnavirus through autophagy. Taken together, our study identified that Avibirnavirus VP3 links PIK3C3-PDPK1 complex to AKT-MTOR pathway and inhibits autophagy, a critical step for controlling virus replication. ABBREVIATIONS: ATG14/Barkor: autophagy related 14; BECN1: beclin 1; CC: coiled-coil; ER: endoplasmic reticulum; hpi: hours post-infection; IBDV: infectious bursal disease virus; IP: co-immunoprecipitation; mAb: monoclonal antibody; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MOI: multiplicity of infection; MTOR: mechanistic target of rapamycin kinase; PDPK1: 3-phosphoinositid-dependent protein kinase-1; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1: sequestosome 1; vBCL2: viral BCL2 apoptosis regulator.

Crystal structure of the tricellulin C-terminal coiled-coil domain reveals a unique mode of dimerization.

Tricellulin is a tight junction protein localized to tricellular contacts in many epithelial tissues, where it is required for full barrier control. Here, we present crystal structures of the tricellulin C-terminal coiled-coil domain, revealing a potential dimeric arrangement. By combining structural, biochemical, functional, and mutation analyses, we gain insight into the mode of tricellulin oligomerization and suggest a model where dimerization of its cytoplasmic C-terminus may play an auxiliary role in stabilizing homophilic and potentially also heterophilic cis-interactions within tight junctions.

Molecular phylogeny and dynamic evolution of disease resistance genes in the legume family.

BACKGROUND: Legumes are the second-most important crop family in agriculture for its economic and nutritional values. Disease resistance (R-) genes play an important role in responding to pathogen infections in plants. To further increase the yield of legume crops, we need a comprehensive understanding of the evolution of R-genes in the legume family. RESULTS: In this study, we developed a robust pipeline and identified a total of 4,217 R-genes in the genomes of seven sequenced legume species. A dramatic diversity of R-genes with structural variances indicated a rapid birth-and-death rate during the R-gene evolution in legumes. The number of R-genes transiently expanded and then quickly contracted after whole-genome duplications, which meant that R-genes were sensitive to subsequent diploidization. R proteins with the Coiled-coil (CC) domain are more conserved than others in legumes. Meanwhile, other types of legume R proteins with only one or two typical domains were subjected to higher rates of loss during evolution. Although R-genes evolved quickly in legumes, they tended to undergo purifying selection instead of positive selection during evolution. In addition, domestication events in some legume species preferentially selected for the genes directly involved in the plant-pathogen interaction pathway while suppressing those R-genes with low occurrence rates. CONCLUSIONS: Our results provide insights into the dynamic evolution of R-genes in the legume family, which will be valuable for facilitating genetic improvements in the disease resistance of legume cultivars.