The Plant Ubiquitin-Proteasome System as a Target for Microbial Manipulation.

The plant immune system perceives pathogens to trigger defense responses. In turn, pathogens secrete effector molecules to subvert these defense responses. The initiation and maintenance of defense responses involve not only de novo synthesis of regulatory proteins and enzymes but also their regulated degradation. The latter is achieved through protein degradation pathways such as the ubiquitin-proteasome system (UPS). The UPS regulates all stages of immunity, from the perception of the pathogen to the execution of the response, and, therefore, constitutes an ideal candidate for microbial manipulation of the host. Pathogen effector molecules interfere with the plant UPS through several mechanisms. This includes hijacking general UPS functions or perturbing its ability to degrade specific targets. In this review, we describe how the UPS regulates different immunity-related processes and how pathogens subvert this to promote disease.

A Pipeline to Monitor Proteasome Homeostasis in Plants.

The proteasome is a key component for regulation of protein turnover across kingdoms. The proteasome has been shown to be involved in or affected by various stress conditions in multiple model organisms in plants. As such, studying proteasome homeostasis is crucial to understand its participation in different cellular conditions. However, the involvement of the proteasome in many cellular processes and its interplay with other degradation pathways hamper the interpretation of experiments based on a single approach. Thus, it is crucial to formulate a framework to investigate proteasome dynamics in different model organisms including plants. Here, we describe a pipeline to monitor proteasome homeostasis using four different methods including (i) luminescent-based proteasome activity measurement, (ii) immunoblot analysis of ubiquitinated proteins, (iii) evaluation of proteasome subunit protein levels, and (iv) monitoring of the proteasome stress regulon on mRNA levels using quantitative real-time PCR (polymerase chain reaction).

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.

Selective autophagy: adding precision in plant immunity.

Plant immunity is antagonized by pathogenic effectors during interactions with bacteria, viruses or oomycetes. These effectors target core plant processes to promote infection. One such core plant process is autophagy, a conserved proteolytic pathway involved in ensuring cellular homeostasis. It involves the formation of autophagosomes around proteins destined for autophagic degradation. Many cellular components from organelles, aggregates, inactive or misfolded proteins have been found to be degraded via autophagy. Increasing evidence points to a high degree of specificity during the targeting of these components, strengthening the idea of selective autophagy. Selective autophagy receptors bridge the gap between target proteins and the forming autophagosome. To achieve this, the receptors are able to recognize specifically their target proteins in a ubiquitin-dependent or -independent manner, and to bind to ATG8 via canonical or non-canonical ATG8-interacting motifs. Some receptors have also been shown to require oligomerization to achieve their function in autophagic degradation. We summarize the recent advances in the role of selective autophagy in plant immunity and highlight NBR1 as a key player. However, not many selective autophagy receptors, especially those functioning in immunity, have been characterized in plants. We propose an in silico approach to identify novel receptors, by screening the Arabidopsis proteome for proteins containing features theoretically needed for a selective autophagy receptor. To corroborate these data, the transcript levels of these proteins during immune response are also investigated using public databases. We further highlight the novel perspectives and applications introduced by immunity-related selective autophagy studies, demonstrating its importance in research.

Robustness of plant quantitative disease resistance is provided by a decentralized immune network.

Quantitative disease resistance (QDR) represents the predominant form of resistance in natural populations and crops. Surprisingly, very limited information exists on the biomolecular network of the signaling machineries underlying this form of plant immunity. This lack of information may result from its complex and quantitative nature. Here, we used an integrative approach including genomics, network reconstruction, and mutational analysis to identify and validate molecular networks that control QDR in Arabidopsis thaliana in response to the bacterial pathogen Xanthomonas campestris To tackle this challenge, we first performed a transcriptomic analysis focused on the early stages of infection and using transgenic lines deregulated for the expression of RKS1, a gene underlying a QTL conferring quantitative and broad-spectrum resistance to XcampestrisRKS1-dependent gene expression was shown to involve multiple cellular activities (signaling, transport, and metabolism processes), mainly distinct from effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses already characterized in Athaliana Protein-protein interaction network reconstitution then revealed a highly interconnected and distributed RKS1-dependent network, organized in five gene modules. Finally, knockout mutants for 41 genes belonging to the different functional modules of the network revealed that 76% of the genes and all gene modules participate partially in RKS1-mediated resistance. However, these functional modules exhibit differential robustness to genetic mutations, indicating that, within the decentralized structure of the QDR network, some modules are more resilient than others. In conclusion, our work sheds light on the complexity of QDR and provides comprehensive understanding of a QDR immune network.

Microbial Effector Proteins - A Journey through the Proteolytic Landscape.

In the evolutionary arms race between pathogens and plants, pathogens evolved effector molecules that they secrete into the host to subvert plant cellular responses in a process termed the effector-targeted pathway (ETP). During recent years the repertoire of ETPs has increased and mounting evidence indicates that the proteasome and autophagy pathways are central hubs of microbial effectors. Both degradation pathways are implicated in a broad array of cellular responses and thus constitute an attractive target for effector proteins to have a broader impact on the host. In this article we first summarize recent findings on how effectors from various pathogens modulate proteolytic pathways and then provide a network analysis of established effector targets implicated in proteolytic degradation machineries. With this network we emphasize the idea that effectors targeting proteolytic degradation pathways will affect the protein synthesis-transport and degradation triangle. We put in perspective that, in utilizing the effector diversity of microbes, we produce excellent tools to study diverse cellular pathways and their possible interplay with each other.