A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component.

Beyond its role in cellular homeostasis, autophagy plays anti- and promicrobial roles in host-microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well-described in animals, the extent to which xenophagy contributes to plant-bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type-III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense-related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense-related autophagy in plant-bacteria interactions.

Salicylic acid and the viral virulence factor 2b regulate the divergent roles of autophagy during cucumber mosaic virus infection.

Macroautophagy/autophagy is a conserved intracellular degradation pathway that has recently emerged as an integral part of plant responses to virus infection. The known mechanisms of autophagy range from the selective degradation of viral components to a more general attenuation of disease symptoms. In addition, several viruses are able to manipulate the autophagy machinery and counteract autophagy-dependent resistance. Despite these findings, the complex interplay of autophagy activities, viral pathogenicity factors, and host defense pathways in disease development remains poorly understood. In the current study, we analyzed the interaction between autophagy and cucumber mosaic virus (CMV) in Arabidopsis thaliana. We show that autophagy is induced during CMV infection and promotes the turnover of the major virulence protein and RNA silencing suppressor 2b. Intriguingly, autophagy induction is mediated by salicylic acid (SA) and dampened by the CMV virulence factor 2b. In accordance with 2b degradation, we found that autophagy provides resistance against CMV by reducing viral RNA accumulation in an RNA silencing-dependent manner. Moreover, autophagy and RNA silencing attenuate while SA promotes CMV disease symptoms, and epistasis analysis suggests that autophagy-dependent disease and resistance are uncoupled. We propose that autophagy counteracts CMV virulence via both 2b degradation and reduced SA-responses, thereby increasing plant fitness with the viral trade-off arising from increased RNA silencing-mediated resistance.

Polycomb Repressive Complex 2 and KRYPTONITE regulate pathogen-induced programmed cell death in Arabidopsis.

The Polycomb Repressive Complex 2 (PRC2) is well-known for its role in controlling developmental transitions by suppressing the premature expression of key developmental regulators. Previous work revealed that PRC2 also controls the onset of senescence, a form of developmental programmed cell death (PCD) in plants. Whether the induction of PCD in response to stress is similarly suppressed by the PRC2 remained largely unknown. In this study, we explored whether PCD triggered in response to immunity- and disease-promoting pathogen effectors is associated with changes in the distribution of the PRC2-mediated histone H3 lysine 27 trimethylation (H3K27me3) modification in Arabidopsis thaliana. We furthermore tested the distribution of the heterochromatic histone mark H3K9me2, which is established, to a large extent, by the H3K9 methyltransferase KRYPTONITE, and occupies chromatin regions generally not targeted by PRC2. We report that effector-induced PCD caused major changes in the distribution of both repressive epigenetic modifications and that both modifications have a regulatory role and impact on the onset of PCD during pathogen infection. Our work highlights that the transition to pathogen-induced PCD is epigenetically controlled, revealing striking similarities to developmental PCD.

Arabidopsis RING-type E3 ubiquitin ligase XBAT35.2 promotes proteasome-dependent degradation of ACD11 to attenuate abiotic stress tolerance.

Plants employ multiple mechanisms to cope with a constantly changing and challenging environment, including using the ubiquitin proteasome system (UPS) to alter their proteome to assist in initiating, modulating and terminating responses to stress. We previously reported that the ubiquitin ligase XBAT35.2 mediates the proteasome-dependent degradation of Accelerated Cell Death 11 (ACD11) to promote pathogen defense. Here, we demonstrate roles for XBAT35.2 and ACD11 in abiotic stress tolerance. As seen in response to pathogen infection, abiotic stress stabilizes XBAT35.2 and the abundance of ACD11 rose consistently with increasing concentrations of abscisic acid (ABA) and salt. Surprisingly, exposure to ABA and salt increased the stability of ACD11, and the overexpression of ACD11 improves plant survival of salt and drought stress, suggesting a role for ACD11 in promoting tolerance. Prolonged exposure to high concentrations of ABA or salt resulted in ubiquitination and the proteasome-dependent degradation of ACD11, however. The stress-induced turnover of ACD11 requires XBAT35.2, as degradation is slowed in the absence of the E3 ubiquitin ligase. Consistent with XBAT35.2 mediating the proteasome-dependent degradation of ACD11, the loss of E3 ubiquitin ligase function enhances the tolerance of salt and drought stress, whereas overexpression increases sensitivity. A model is presented where, upon the perception of abiotic stress, ACD11 abundance increases to promote tolerance. Meanwhile, XBAT35.2 accumulates and in turn promotes the degradation of ACD11 to attenuate the stress response. The results characterize XBAT35.2 as an E3 ubiquitin ligase with opposing roles in abiotic and biotic stress.

Autophagy-virus interplay in plants: from antiviral recognition to proviral manipulation.

Autophagy is a conserved self-cleaning and renewal system required for cellular homeostasis and stress tolerance. Autophagic processes are also implicated in the response to 'non-self' such as viral pathogens, yet the functions and mechanisms of autophagy during plant virus infection have only recently started to be revealed. Compelling evidence now indicates that autophagy is an integral part of antiviral immunity in plants. It can promote the hypersensitive cell death response upon incompatible viral infections or mediate the selective elimination of entire particles and individual proteins from compatible viruses in a pathway similar to xenophagy in animals. Several viruses, however, have evolved measures to antagonize xenophagic degradation or utilize autophagy to suppress disease-associated cell death and other defence pathways like RNA silencing. Here, we highlight the current advances and gaps in our understanding of the complex autophagy-virus interplay and its consequences for host immunity and viral pathogenesis in plants.

Anti- and pro-microbial roles of autophagy in plant-bacteria interactions.

Macroautophagy/autophagy and the ubiquitin-proteasome system (UPS) are major proteolytic pathways that are increasingly recognized as battlegrounds during host-microbe interactions in eukaryotes. In plants, the UPS has emerged as central component of innate immunity and is manipulated by bacterial pathogens to enhance virulence. Autophagy has been ascribed a similar importance for anti-bacterial immunity in animals, but the contribution of autophagy to host-bacteria interactions remained elusive in plants. Here, we present and discuss our recent findings that revealed anti- and pro-bacterial roles of autophagy pathways during bacterial infection in the model plant Arabidopsis thaliana. We discovered that selective autophagy mediated by the autophagy cargo receptor AT4G24690/NBR1 limits growth of Pseudomonas syringae pv. tomato DC3000 (Pst) by suppressing the establishment of an aqueous extracellular space ('water-soaking'). In turn, Pseudomonas employs the effector protein HopM1 to activate autophagy and proteasome degradation ('proteaphagy'), thereby enhancing its pathogenicity. Thus, our study demonstrates that distinct selective autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between the proteasome and autophagy system in plant-bacterial interactions.

Bacteria Exploit Autophagy for Proteasome Degradation and Enhanced Virulence in Plants.

Autophagy and the ubiquitin-proteasome system (UPS) are two major protein degradation pathways implicated in the response to microbial infections in eukaryotes. In animals, the contribution of autophagy and the UPS to antibacterial immunity is well documented and several bacteria have evolved measures to target and exploit these systems to the benefit of infection. In plants, the UPS has been established as a hub for immune responses and is targeted by bacteria to enhance virulence. However, the role of autophagy during plant-bacterial interactions is less understood. Here, we have identified both pro- and antibacterial functions of autophagy mechanisms upon infection of Arabidopsis thaliana with virulent Pseudomonas syringae pv tomato DC3000 (Pst). We show that Pst activates autophagy in a type III effector (T3E)-dependent manner and stimulates the autophagic removal of proteasomes (proteaphagy) to support bacterial proliferation. We further identify the T3E Hrp outer protein M1 (HopM1) as a principle mediator of autophagy-inducing activities during infection. In contrast to the probacterial effects of Pst-induced proteaphagy, NEIGHBOR OF BRCA1-dependent selective autophagy counteracts disease progression and limits the formation of HopM1-mediated water-soaked lesions. Together, we demonstrate that distinct autophagy pathways contribute to host immunity and bacterial pathogenesis during Pst infection and provide evidence for an intimate crosstalk between proteasome and autophagy in plant-bacterial interactions.

Autophagy-related approaches for improving nutrient use efficiency and crop yield protection.

Autophagy is a eukaryotic catabolic pathway essential for growth and development. In plants, it is activated in response to environmental cues or developmental stimuli. However, in contrast to other eukaryotic systems, we know relatively little regarding the molecular players involved in autophagy and the regulation of this complex pathway. In the framework of the COST (European Cooperation in Science and Technology) action TRANSAUTOPHAGY (2016-2020), we decided to review our current knowledge of autophagy responses in higher plants, with emphasis on knowledge gaps. We also assess here the potential of translating the acquired knowledge to improve crop plant growth and development in a context of growing social and environmental challenges for agriculture in the near future.

Transcriptional stimulation of rate-limiting components of the autophagic pathway improves plant fitness.

Autophagy is a major catabolic process whereby autophagosomes deliver cytoplasmic content to the lytic compartment for recycling. Autophagosome formation requires two ubiquitin-like systems conjugating Atg12 with Atg5, and Atg8 with lipid phosphatidylethanolamine (PE), respectively. Genetic suppression of these systems causes autophagy-deficient phenotypes with reduced fitness and longevity. We show that Atg5 and the E1-like enzyme, Atg7, are rate-limiting components of Atg8-PE conjugation in Arabidopsis. Overexpression of ATG5 or ATG7 stimulates Atg8 lipidation, autophagosome formation, and autophagic flux. It also induces transcriptional changes opposite to those observed in atg5 and atg7 mutants, favoring stress resistance and growth. As a result, ATG5- or ATG7-overexpressing plants exhibit increased resistance to necrotrophic pathogens and oxidative stress, delayed aging and enhanced growth, seed set, and seed oil content. This work provides an experimental paradigm and mechanistic insight into genetic stimulation of autophagy in planta and shows its efficiency for improving plant productivity.

Turnip Mosaic Virus Counteracts Selective Autophagy of the Viral Silencing Suppressor HCpro.

Autophagy is a conserved intracellular degradation pathway and has emerged as a key mechanism of antiviral immunity in metazoans, including the selective elimination of viral components. In turn, some animal viruses are able to escape and modulate autophagy for enhanced pathogenicity. Whether host autophagic responses and viral countermeasures play similar roles in plant-virus interactions is not well understood. Here, we have identified selective autophagy as antiviral pathway during plant infection with turnip mosaic virus (TuMV), a positive-stranded RNA potyvirus. We show that the autophagy cargo receptor NBR1 suppresses viral accumulation by targeting the viral RNA silencing suppressor helper-component proteinase (HCpro), presumably in association with virus-induced RNA granules. Intriguingly, TuMV seems to antagonize NBR1-dependent autophagy during infection by the activity of distinct viral proteins, thereby limiting its antiviral capacity. We also found that NBR1-independent bulk autophagy prevents premature plant death, thus extending the lifespan of virus reservoirs and particle production. Together, our study highlights a conserved role of selective autophagy in antiviral immunity and suggests the evolvement of viral protein functions to inhibit autophagy processes, despite a potential trade-off in host survival.

Vacuole Integrity Maintained by DUF300 Proteins Is Required for Brassinosteroid Signaling Regulation.

Brassinosteroid (BR) hormone signaling controls multiple processes during plant growth and development and is initiated at the plasma membrane through the receptor kinase BRASSINOSTEROID INSENSITIVE1 (BRI1) together with co-receptors such as BRI1-ASSOCIATED RECEPTOR KINASE1 (BAK1). BRI1 abundance is regulated by endosomal recycling and vacuolar targeting, but the role of vacuole-related proteins in BR receptor dynamics and BR responses remains elusive. Here, we show that the absence of two DUF300 domain-containing tonoplast proteins, LAZARUS1 (LAZ1) and LAZ1 HOMOLOG1 (LAZ1H1), causes vacuole morphology defects, growth inhibition, and constitutive activation of BR signaling. Intriguingly, tonoplast accumulation of BAK1 was substantially increased and appeared causally linked to enhanced BRI1 trafficking and degradation in laz1 laz1h1 plants. Since unrelated vacuole mutants exhibited normal BR responses, our findings indicate that DUF300 proteins play distinct roles in the regulation of BR signaling by maintaining vacuole integrity required to balance subcellular BAK1 pools and BR receptor distribution.

NBR1-mediated antiviral xenophagy in plant immunity.

Macroautophagy/autophagy intersects with metazoan virus infections in highly complex and multifaceted ways. Autophagy mechanisms are part of antiviral immunity, but can be manipulated by several viruses to the benefit of infection. In plants, however, the roles of autophagy in virus infections have only recently started to emerge. Here, we present and discuss our recent study that identified 2 prominent functions of autophagy upon cauliflower mosaic virus (CaMV) infection in Arabidopsis. We found that "bulk" autophagy significantly extended the life span of infected plants and increased total virus production. In addition to this proviral role, we discovered that the selective autophagy receptor protein AT4G24690/NBR1 binds viral particles to mediate their xenophagic degradation. Intriguingly, CaMV inclusion bodies protect viral particles from xenophagy and thus represent a sophisticated strategy to counter the antiviral capacity while maintaining the proviral activity of autophagy. Together, our study gives a seminal description of how autophagy is integrated into host immunity and viral pathogenesis in plants, and provides a primary example for removal of a plant pathogen by xenophagy.

The RING-Type E3 Ligase XBAT35.2 Is Involved in Cell Death Induction and Pathogen Response.

XBAT35 belongs to a subfamily of Arabidopsis (Arabidopsis thaliana) RING-type E3s that are similar in domain architecture to the rice (Oryza sativa) XA21 Binding Protein3, a defense protein. The XBAT35 transcript undergoes alternative splicing to produce two protein isoforms, XBAT35.1 and XBAT35.2. Here, we demonstrate that XBAT35.2 localizes predominantly to the Golgi and is involved in cell death induction and pathogen response. XBAT35.2, but not XBAT35.1, was found to trigger cell death when overexpressed in tobacco (Nicotiana benthamiana) leaves and does so in a manner that requires its RING domain. Loss of XBAT35 gene function disrupts the plant's ability to defend against pathogen attack, whereas overexpression of XBAT35.2 enhances resistance to pathogens. XBAT35.2 was found to be unstable and promotes its own degradation, suggesting self-regulation. Inoculation with virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv tomato DC3000 results in a drastic reduction in the levels of ubiquitinated XBAT35.2 and an increase in the abundance of the E3. This implies that pathogen infection prohibits XBAT35.2 self-regulation and stabilizes the E3. In agreement with a role in defending against pathogens, XBAT35.2 interacts with defense-related Accelerated Cell Death11 (ACD11) in planta and promotes the proteasome-dependent turnover of ACD11 in cell-free degradation assays. In accordance with regulation by a stabilized XBAT35.2, the levels of ubiquitinated ACD11 increased considerably, and the abundance of ACD11 was reduced following pathogen infection. In addition, treatment of transgenic seedlings with a proteasome inhibitor results in the accumulation of ACD11, confirming proteasome-dependent degradation. Collectively, these results highlight a novel role for XBAT35.2 in cell death induction and defense against pathogens.

Autophagy as a mediator of life and death in plants.

Autophagy is a major pathway for degradation and recycling of cytoplasmic material, including individual proteins, aggregates, and entire organelles. Autophagic processes serve mainly survival functions in cellular homeostasis, stress adaptation and immune responses but can also have death-promoting activities in different eukaryotic organisms. In plants, the role of autophagy in the regulation of programmed cell death (PCD) remained elusive and a subject of debate. More recent evidence, however, has resulted in the consensus that autophagy can either promote or restrict different forms of PCD. Here, we present latest advances in understanding the molecular mechanisms and functions of plant autophagy and discuss their implications for life and death decisions in the context of developmental and pathogen-induced PCD.

Autophagy as an emerging arena for plant-pathogen interactions.

Autophagy is a highly conserved degradation and recycling process that controls cellular homeostasis, stress adaptation, and programmed cell death in eukaryotes. Emerging evidence indicates that autophagy is a key regulator of plant innate immunity and contributes with both pro-death and pro-survival functions to antimicrobial defences, depending on the pathogenic lifestyle. In turn, several pathogens have co-opted and evolved strategies to manipulate host autophagy pathways to the benefit of infection, while some eukaryotic microbes require their own autophagy machinery for successful pathogenesis. In this review, we present and discuss recent advances that exemplify the important role of pro- and antimicrobial autophagy in plant-pathogen interactions.

Salicylic acid interferes with GFP fluorescence in vivo.

Fluorescent proteins have become essential tools for cell biologists. They are routinely used by plant biologists for protein and promoter fusions to infer protein localization, tissue-specific expression and protein abundance. When studying the effects of biotic stress on chromatin, we unexpectedly observed a decrease in GFP signal intensity upon salicylic acid (SA) treatment in Arabidopsis lines expressing histone H1-GFP fusions. This GFP signal decrease was dependent on SA concentration. The effect was not specific to the linker histone H1-GFP fusion but was also observed for the nucleosomal histone H2A-GFP fusion. This result prompted us to investigate a collection of fusion proteins, which included different promoters, subcellular localizations and fluorophores. In all cases, fluorescence signals declined strongly or disappeared after SA application. No changes were detected in GFP-fusion protein abundance when fluorescence signals were lost indicating that SA does not interfere with protein stability but GFP fluorescence. In vitro experiments showed that SA caused GFP fluorescence reduction only in vivo but not in vitro, suggesting that SA requires cellular components to cause fluorescence reduction. Together, we conclude that SA can interfere with the fluorescence of various GFP-derived reporter constructs in vivo. Assays that measure relocation or turnover of GFP-tagged proteins upon SA treatment should therefore be evaluated with caution.

Selective autophagy limits cauliflower mosaic virus infection by NBR1-mediated targeting of viral capsid protein and particles.

Autophagy plays a paramount role in mammalian antiviral immunity including direct targeting of viruses and their individual components, and many viruses have evolved measures to antagonize or even exploit autophagy mechanisms for the benefit of infection. In plants, however, the functions of autophagy in host immunity and viral pathogenesis are poorly understood. In this study, we have identified both anti- and proviral roles of autophagy in the compatible interaction of cauliflower mosaic virus (CaMV), a double-stranded DNA pararetrovirus, with the model plant Arabidopsis thaliana We show that the autophagy cargo receptor NEIGHBOR OF BRCA1 (NBR1) targets nonassembled and virus particle-forming capsid proteins to mediate their autophagy-dependent degradation, thereby restricting the establishment of CaMV infection. Intriguingly, the CaMV-induced virus factory inclusions seem to protect against autophagic destruction by sequestering capsid proteins and coordinating particle assembly and storage. In addition, we found that virus-triggered autophagy prevents extensive senescence and tissue death of infected plants in a largely NBR1-independent manner. This survival function significantly extends the timespan of virus production, thereby increasing the chances for virus particle acquisition by aphid vectors and CaMV transmission. Together, our results provide evidence for the integration of selective autophagy into plant immunity against viruses and reveal potential viral strategies to evade and adapt autophagic processes for successful pathogenesis.

Retromer contributes to immunity-associated cell death in Arabidopsis.

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.

Chromatin assembly factor CAF-1 represses priming of plant defence response genes.

Plants have evolved efficient defence systems against pathogens that often rely on specific transcriptional responses. Priming is part of the defence syndrome, by establishing a hypersensitive state of defence genes such as after a first encounter with a pathogen. Because activation of defence responses has a fitness cost, priming must be tightly controlled to prevent spurious activation of defence. However, mechanisms that repress defence gene priming are poorly understood. Here, we show that the histone chaperone CAF-1 is required to establish a repressed chromatin state at defence genes. Absence of CAF-1 results in spurious activation of a salicylic acid-dependent pathogen defence response in plants grown under non-sterile conditions. Chromatin at defence response genes in CAF-1 mutants under non-inductive (sterile) conditions is marked by low nucleosome occupancy and high H3K4me3 at transcription start sites, resembling chromatin in primed wild-type plants. We conclude that CAF-1-mediated chromatin assembly prevents the establishment of a primed state that may under standard non-sterile growth conditions result in spurious activation of SA-dependent defence responses and consequential reduction of plant vigour.