EXTRA LARGE G-PROTEIN2 mediates cell death and hyperimmunity in the chitin elicitor receptor kinase 1-4 mutant.

Heterotrimeric G-proteins are signal transduction complexes that comprised three subunits, Gα, Gß, and Gγ, and are involved in many aspects of plant life. The noncanonical Gα subunit EXTRA LARGE G-PROTEIN2 (XLG2) mediates pathogen-associated molecular pattern (PAMP)-induced reactive oxygen species (ROS) generation and immunity downstream of pattern recognition receptors. A mutant of the chitin receptor component CHITIN ELICITOR RECEPTOR KINASE1 (CERK1), cerk1-4, maintains normal chitin signaling capacity but shows excessive cell death upon infection with powdery mildew fungi. We identified XLG2 mutants as suppressors of the cerk1-4 phenotype. Mutations in XLG2 complex partners ARABIDOPSIS Gß1 (AGB1) and Gγ1 (AGG1) have a partial cerk1-4 suppressor effect. Contrary to its role in PAMP-induced immunity, XLG2-mediated control of ROS production by RESPIRATORY BURST OXIDASE HOMOLOGUE D (RBOHD) is not critical for cerk1-4-associated cell death and hyperimmunity. The cerk1-4 phenotype is also independent of the co-receptor/adapter kinases BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1) and SUPPRESSOR OF BIR1 1 (SOBIR1), but requires the E3 ubiquitin ligase PLANT U-BOX 2 (PUB2). XLG2 localizes to both the cell periphery and nucleus, and the cerk1-4 cell death phenotype is mediated by the cell periphery pool of XLG2. Integrity of the XLG2 N-terminal domain, but not its phosphorylation, is essential for correct XLG2 localization and formation of the cerk1-4 phenotype. Our results support a model in which XLG2 acts downstream of an unknown cell surface receptor that activates an NADPH oxidase-independent cell death pathway in Arabidopsis (Arabidopsis thaliana).

The Leaf Microbiome of Arabidopsis Displays Reproducible Dynamics and Patterns throughout the Growing Season.

Leaves are primarily responsible for the plant's photosynthetic activity. Thus, changes in the leaf microbiota, which includes deleterious and beneficial microbes, can have far-reaching effects on plant fitness and productivity. Identifying the processes and microorganisms that drive these changes over a plant's lifetime is, therefore, crucial. In this study, we analyzed the temporal dynamics in the leaf microbiome of Arabidopsis thaliana, integrating changes in both composition and microbe-microbe interactions via the study of microbial networks. Field-grown Arabidopsis were used to monitor leaf bacterial, fungal and oomycete communities throughout the plant's natural growing season (extending from November to March) over three consecutive years. Our results revealed the existence of conserved temporal patterns, with microbial communities and networks going through a stabilization phase of decreased diversity and variability at the beginning of the plant's growing season. Despite a high turnover in these communities, we identified 19 "core" taxa persisting on Arabidopsis leaves across time and plant generations. With the hypothesis these microbes could be playing key roles in the structuring of leaf microbial communities, we conducted a time-informed microbial network analysis which showed core taxa are not necessarily highly connected network "hubs," and "hubs" alternate with time. Our study shows that leaf microbial communities exhibit reproducible dynamics and patterns, suggesting the potential of using our understanding of temporal trajectories in microbial community composition to design experiments aimed at driving these communities toward desired states. IMPORTANCE Utilizing plant microbiota to promote plant growth and plant health is key to more environmentally friendly agriculture. A major bottleneck in the engineering of plant-beneficial microbial communities is the low persistence of applied microbes under filed conditions, especially considering plant leaves. Indeed, although many leaf-associated microorganisms have the potential to promote plant growth and protect plants from pathogens, few of them are able to survive and thrive over time. In our study, we could show that leaf microbial communities are very variable at the beginning of the plant growing season but become more and more similar and less variable as the season progresses. We further identify a cohort of 19 "core" microbes, systematically present on plant leaves that would make these microbes exceptional candidates for future agricultural applications.

A fungal member of the Arabidopsis thaliana phyllosphere antagonizes Albugo laibachii via a GH25 lysozyme.

Plants are not only challenged by pathogenic organisms but also colonized by commensal microbes. The network of interactions these microbes establish with their host and among each other is suggested to contribute to the immune responses of plants against pathogens. In wild Arabidopsis thaliana populations, the oomycete pathogen Albugo laibachii plays an influential role in structuring the leaf phyllosphere. We show that the epiphytic yeast Moesziomyces bullatus ex Albugo on Arabidopsis, a close relative of pathogenic smut fungi, is an antagonistic member of the A. thaliana phyllosphere, which reduces infection of A. thaliana by A. laibachii. Combination of transcriptomics, reverse genetics, and protein characterization identified a GH25 hydrolase with lysozyme activity as a major effector of this microbial antagonism. Our findings broaden the understanding of microbial interactions within the phyllosphere, provide insights into the evolution of epiphytic basidiomycete yeasts, and pave the way for novel biocontrol strategies.

Much like the 'good bacteria' that live in our guts, many microscopic organisms can co-exist with and even benefit the plants they live on. For instance, the yeast Moesziomyces bullatus ex Albugo (MbA for short) can shield the leaves of its plant host against white rust, a disease caused by the organism Albugo laibachii. Studies have started to unveil how the various microbes at the surface of leaves interact and regulate their own community, yet the genetic mechanisms at play are less well-known. To investigate these processes, Eitzen et al. examined the genes that were switched on when MbA cells were in contact with A. laibachii on a leaf. This experiment revealed a few gene candidates that were then deleted, one by one, in MbA cells. As a result, a gene emerged as being key to protect the plant from white rust. It produces an enzyme known as the GH25 hydrolase, which, when purified, could reduce A. laibachii infections on plant leaves. Bacteria, fungi and other related microorganisms cause many diseases which, like white rust, can severely affect crops. Chemical methods exist to prevent these infections but they can have many biological and ecological side effects. A solution inspired by natural interactions may be safer and more effective at managing plant diseases that affect valuable crops. Harnessing the interactions between microbes living on plants, and the GH25 enzyme, may offer better disease control.

Genomic dissection of host-microbe and microbe-microbe interactions for advanced plant breeding.

Agriculture faces many emerging challenges to sustainability, including limited nutrient resources, losses from diseases caused by current and emerging pathogens and environmental degradation. Microorganisms have great importance for plant growth and performance, including the potential to increase yields, nutrient uptake and pathogen resistance. An urgent need is therefore to understand and engineer plants and their associated microbial communities. Recent massive genomic sequencing of host plants and associated microbes offers resources to identify novel mechanisms of communal assembly mediated by the host. For example, host-microbe and microbe-microbe interactions are involved in niche formation, thereby contributing to colonization. By leveraging genomic resources, genetic traits underlying those mechanisms will become important resources to design plants selecting and hosting beneficial microbial communities.

Microbial Hub Taxa Link Host and Abiotic Factors to Plant Microbiome Variation.

Plant-associated microorganisms have been shown to critically affect host physiology and performance, suggesting that evolution and ecology of plants and animals can only be understood in a holobiont (host and its associated organisms) context. Host-associated microbial community structures are affected by abiotic and host factors, and increased attention is given to the role of the microbiome in interactions such as pathogen inhibition. However, little is known about how these factors act on the microbial community, and especially what role microbe-microbe interaction dynamics play. We have begun to address this knowledge gap for phyllosphere microbiomes of plants by simultaneously studying three major groups of Arabidopsis thaliana symbionts (bacteria, fungi and oomycetes) using a systems biology approach. We evaluated multiple potential factors of microbial community control: we sampled various wild A. thaliana populations at different times, performed field plantings with different host genotypes, and implemented successive host colonization experiments under lab conditions where abiotic factors, host genotype, and pathogen colonization was manipulated. Our results indicate that both abiotic factors and host genotype interact to affect plant colonization by all three groups of microbes. Considering microbe-microbe interactions, however, uncovered a network of interkingdom interactions with significant contributions to community structure. As in other scale-free networks, a small number of taxa, which we call microbial "hubs," are strongly interconnected and have a severe effect on communities. By documenting these microbe-microbe interactions, we uncover an important mechanism explaining how abiotic factors and host genotypic signatures control microbial communities. In short, they act directly on "hub" microbes, which, via microbe-microbe interactions, transmit the effects to the microbial community. We analyzed two "hub" microbes (the obligate biotrophic oomycete pathogen Albugo and the basidiomycete yeast fungus Dioszegia) more closely. Albugo had strong effects on epiphytic and endophytic bacterial colonization. Specifically, alpha diversity decreased and beta diversity stabilized in the presence of Albugo infection, whereas they otherwise varied between plants. Dioszegia, on the other hand, provided evidence for direct hub interaction with phyllosphere bacteria. The identification of microbial "hubs" and their importance in phyllosphere microbiome structuring has crucial implications for plant-pathogen and microbe-microbe research and opens new entry points for ecosystem management and future targeted biocontrol. The revelation that effects can cascade through communities via "hub" microbes is important to understand community structure perturbations in parallel fields including human microbiomes and bioprocesses. In particular, parallels to human microbiome "keystone" pathogens and microbes open new avenues of interdisciplinary research that promise to better our understanding of functions of host-associated microbiomes.