Subversion of selective autophagy for the biogenesis of tombusvirus replication organelles inhibits autophagy.

Elaborate viral replication organelles (VROs) are formed to support positive-strand RNA virus replication in infected cells. VRO formation requires subversion of intracellular membranes by viral replication proteins. Here, we showed that the key ATG8f autophagy protein and NBR1 selective autophagy receptor were co-opted by Tomato bushy stunt virus (TBSV) and the closely-related carnation Italian ringspot virus. Knockdown of ATG8f or NBR1 in plants led to reduced tombusvirus replication, suggesting pro-viral function for selective autophagy. BiFC and proximity-labeling experiments showed that the TBSV p33 replication protein interacted with ATG8f and NBR1 to recruit them to VROs. In addition, we observed that several core autophagy proteins, such as ATG1a, ATG4, ATG5, ATG101 and the plant-specific SH3P2 autophagy adaptor proteins were also re-localized to TBSV VROs, suggesting that TBSV hijacks the autophagy machinery in plant cells. We demonstrated that subversion of autophagy components facilitated the recruitment of VPS34 PI3 kinase and enrichment of phospholipids, such as phosphatidylethanolamine and PI3P phosphoinositide in the VRO membranes. Hijacking of autophagy components into TBSV VROs led to inhibition of autophagic flux. We also found that a fraction of the subverted ATG8f and NBR1 was sequestered in biomolecular condensates associated with VROs. We propose that the VRO-associated condensates trap those autophagy proteins, taking them away from the autophagy pathway. Overall, tombusviruses hijack selective autophagy to provide phospholipid-rich membranes for replication and to regulate the antiviral autophagic flux.

ENHANCED DISEASE SUSCEPTIBILITY 1 promotes hydrogen peroxide scavenging to enhance rice thermotolerance.

Heat stress is a major factor limiting the production and geographic distribution of rice (Oryza sativa), and breeding rice varieties with tolerance to heat stress is of immense importance. Although extensive studies have revealed that reactive oxygen species (ROS) play a critical role in rice acclimation to heat stress, the molecular basis of rice controlling ROS homeostasis remains largely unclear. In this study, we discovered a novel heat-stress-responsive strategy that orchestrates ROS homeostasis centering on an immune activator, rice ENHANCED DISEASE SUSCEPTIBILITY 1 (OsEDS1). OsEDS1, which confers heat stress tolerance, promotes hydrogen peroxide (H2O2) scavenging by stimulating catalase activity through the OsEDS1-catalase association. The loss-of-function mutation in OsEDS1 causes increased sensitivity to heat stress, whereas the overexpression of OsEDS1 enhances thermotolerance. Furthermore, overexpression lines greatly improved rice tolerance to heat stress during the reproductive stage, which was associated with substantially increased seed setting, grain weight, and plant yield. Rice CATALASE C (OsCATC), whose activity is promoted by OsEDS1, degrades H2O2 to activate rice heat stress tolerance. Our findings greatly expand our understanding of heat stress responses in rice. We reveal a molecular framework that promotes heat tolerance through ROS homeostasis regulation, suggesting a theoretical basis and providing genetic resources for breeding heat-tolerant rice varieties.

Key tethering function of Atg11 autophagy scaffold protein in formation of virus-induced membrane contact sites during tombusvirus replication.

Positive-strand RNA viruses induce the biogenesis of viral replication organelles (VROs), which support viral replication in infected cells. VRO formation requires viral replication proteins, co-opted host factors and intracellular membranes. Here, we show that the conserved Atg11 autophagy scaffold protein is co-opted by Tomato bushy stunt virus (TBSV) via direct interactions with the viral replication proteins. Deletion of ATG11 in yeast or knockdown of the homologous Atg11 in plants led to reduced tombusvirus replication, thus indicating pro-viral function for Atg11. Based on co-purification, BiFC and proximity-labeling experiments, we find that Atg11 is co-opted to stabilize virus-induced membrane contact sites (vMCS) within VROs. We propose that the tethering and scaffold function of Atg11 is critical in vMCSs for lipid enrichment. Absence of Atg11 interferes with sterols enrichment in VROs, rendering VROs RNAi-sensitive. Altogether, the expanding roles of co-opted host proteins with tethering functions suggest that the tombusvirus VROs are elaborate structures.

Two VQ Proteins are Substrates of the OsMPKK6-OsMPK4 Cascade in Rice Defense Against Bacterial Blight.

BACKGROUND: The plant-specific valine-glutamine (VQ) protein family with the conserved motif FxxxVQxLTG reportedly functions with the mitogen-activated protein kinase (MAPK) in plant immunity. However, the roles of VQ proteins in MAPK-mediated resistance to disease in rice remain largely unknown. RESULTS: In this study, two rice VQ proteins OsVQ14 and OsVQ32 were newly identified to function as the signaling components of a MAPK cascade, OsMPKK6-OsMPK4, to regulate rice resistance to Xanthomonas oryzae pv. oryzae (Xoo). Both OsVQ14 and OsVQ32 positively regulated rice resistance to Xoo. In vitro and in vivo studies revealed that OsVQ14 and OsVQ32 physically interacted with and were phosphorylated by OsMPK4. OsMPK4 was highly phosphorylated in transgenic plants overexpressing OsMPKK6, which showed enhanced resistance to Xoo. Meanwhile, phosphorylated OsVQ14 and OsVQ32 were also markedly accumulated in OsMPKK6-overexpressing transgenic plants. CONCLUSIONS: We discovered that OsVQ14 and OsVQ32 functioned as substrates of the OsMPKK6-OsMPK4 cascade to enhance rice resistance to Xoo, thereby defining a more complete signal transduction pathway for induced defenses.

Jasmonic Acid-Involved OsEDS1 Signaling in Rice-Bacteria Interactions.

BACKGROUND: The function of Arabidopsis enhanced disease susceptibility 1 (AtEDS1) and its sequence homologs in other dicots have been extensively studied. However, it is unknown whether rice EDS1 homolog (OsEDS1) plays a role in regulating the rice-pathogen interaction. RESULTS: In this study, a OsEDS1-knouckout mutant (oseds1) was characterized and shown to have increased susceptibility to Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), suggesting the positive role of OsEDS1 in regulating rice disease resistance. However, the following evidence suggests that OsEDS1 shares some differences with AtEDS1 in its way to regulate the host-pathogen interactions. Firstly, OsEDS1 modulates the rice-bacteria interactions involving in jasmonic acid (JA) signaling pathway, while AtEDS1 regulates Arabidopsis disease resistance against biotrophic pathogens depending on salicylic acid (SA) signaling pathway. Secondly, introducing AtEDS1 could reduce oseds1 mutant susceptibility to Xoo rather than to Xoc. Thirdly, exogenous application of JA and SA cannot complement the susceptible phenotype of the oseds1 mutant, while exogenous application of SA is capable of complementing the susceptible phenotype of the ateds1 mutant. Finally, OsEDS1 is not required for R gene mediated resistance, while AtEDS1 is required for disease resistance mediated by TIR-NB-LRR class of R proteins. CONCLUSION: OsEDS1 is a positive regulator in rice-pathogen interactions, and shares both similarities and differences with AtEDS1 in its way to regulate plant-pathogen interactions.