Biology subjects

Jones, A. M. E.

Publications and source records attributed to Jones, A. M. E..

4 recordsLinked to original sources

bioRxiv2021

Meta-exoproteomics identifies active plant-microbe interactions operating in the rhizosphere

The advance of DNA sequencing technologies has drastically changed our perception of the complexity and structure of the plant microbiome and its role in augmenting plant health. By comparison, our ability to accurately identify the metabolically active fraction of soil microbiota and their specific functional role is relatively limited. Here, we combined our recently developed protein extraction method and an iterative bioinformatics pipeline to enable the capture and identification of extracellular proteins (meta-exoproteomics) expressed in the rhizosphere of Brassica spp. First, we validated our method in the laboratory by successfully identifying proteins related to the host plant (Brassica rapa) and a bacterial inoculant, Pseudomonas putida BIRD-1, revealing the latter expressed numerous rhizosphere specific proteins linked to the acquisition of plant-derived nutrients. Next, we analysed natural field-soil microbial communities associated with Brassica napus L (Oil Seed rape). By combining deep-sequencing metagenomics with meta-exoproteomics, a total of 1882 proteins were identified in bulk and rhizosphere samples. Importantly, meta-exoproteomics identified a clear shift (p<0.001) in the metabolically active fraction of the soil microbiota responding to the presence of B. napus roots that was not apparent in the composition of the total microbial community (metagenome). This metabolic shift was associated with the stimulation of rhizosphere-specialised bacteria, such as Gammaproteobacteria, Betaproteobacteria and Flavobacteriia and the upregulation of plant beneficial functions related to phosphorus and nitrogen mineralisation. By providing the first meta-proteomic level assessment of the active plant microbiome at the field-scale, this study demonstrates the importance of moving past a genomic assessment of the plant microbiome in order to determine ecologically important plant: microbe interactions driving plant growth.

microbiology↗

bioRxiv2021

ACTIVATION LOOP PHOSPHORYLATION OF A NON-RD RECEPTOR KINASE INITIATES PLANT INNATE IMMUNE SIGNALING

Receptor kinases (RKs) play fundamental roles in extracellular sensing to regulate development and stress responses across kingdoms. In plants, leucine-rich repeat receptor kinases (LRR-RKs) function primarily as peptide receptors that regulate myriad aspects of plant development and response to external stimuli. Extensive phosphorylation of LRR-RK cytoplasmic domains is among the earliest detectable responses following ligand perception, and reciprocal transphosphorylation between a receptor and its co-receptor is thought to activate the receptor complex. Originally proposed based on characterization of the brassinosteroid receptor, the prevalence of complex activation via reciprocal transphosphorylation across the plant RK family has not been tested. Using the LRR-RK ELONGATION FACTOR TU RECEPTOR (EFR) as a model RK, we set out to understand the steps critical for activating RK complexes. While the EFR cytoplasmic domain is an active protein kinase in vitro and is phosphorylated in a ligand-dependent manner in vivo, catalytically deficient EFR variants are functional in anti-bacterial immunity. These results reveal a non-catalytic role for the EFR cytoplasmic domain in triggering immune signaling and indicate that reciprocal transphoshorylation is not a ubiquitous requirement for LRR-RK complex activation. Rather, our analysis of EFR along with a detailed survey of the literature suggests a distinction between LRR-RK complexes with RD- versus non-RD protein kinase domains. Based on newly identified phosphorylation sites that regulate the activation state of the EFR complex in vivo, we propose that LRR-RK complexes containing a non-RD protein kinase may be regulated by phosphorylation-dependent conformational changes of the ligand-binding receptor which could initiate signaling in a feed-forward fashion either allosterically or through driving the dissociation of negative regulators of the complex.

biochemistry↗

bioRxiv2020

Structural and functional insights into the mechanism of action of plant boron transporters

Boron has essential roles in plant growth and development. BOR proteins are key in the active uptake and distribution of boron, and regulation of intracellular boron concentrations. However, their mechanism of action remains poorly studied. BOR proteins are members of the SLC4 family of transporters and thus homologues of well studied mammalian transporters including the human Anion Exchanger 1 (hAE1). Here we generated Arabidopsis thaliana BOR1 (AtBOR1) variants based i) on known disease causing mutations of hAE1 (S466R, A500R) and ii) a loss of function mutation (D311A) identified in the yeast BOR protein, ScBOR1p. The AtBOR1 variants express in yeast and localise to the plasma membrane, although both S466R and A500R exhibit lower expression than the WT AtBOR1 and D311A. The D311A, S466R and A500R mutations result in a loss of boron efflux activity in a yeast bor1p knockout strain. A. thaliana plants containing these three individual mutations exhibit substantially decreased growth phenotypes in soil under conditions of low boron. These data confirm an important role for D311 in the function of the protein and show that mutations equivalent to disease causing mutations in hAE1 have major effects in AtBOR1. We also obtained a low resolution cryo-EM structure of a BOR protein from Oryza sativa, OsBOR3 lacking the 30 C-terminal amino acids. This structure confirms the gate and core domain organisation previously observed for related proteins, and is strongly suggestive of an inward facing conformation.

plant biology↗

bioRxiv2020

Host-interactor screens of Phytophthora infestans RXLR proteins reveal vesicle trafficking as a major effector-targeted process

Pathogens modulate plant cell structure and function by secreting effectors into host tissues. Effectors typically function by associating with host molecules and modulating their activities. This study aimed to identify the host processes targeted by the RXLR class of host-translocated effectors of the potato blight pathogen Phytophthora infestans. To this end, we performed an in planta protein-protein interaction screen by transiently expressing P. infestans RXLR effectors in Nicotiana benthamiana leaves followed by co-immunoprecipitation (co-IP) and liquid chromatography tandem mass spectrometry (LC-MS/MS). This screen generated an effector-host protein interactome matrix of 59 P. infestans RXLR effectors x 586 N. benthamiana proteins. Classification of the host interactors into putative functional categories revealed over 35 biological processes possibly targeted by P. infestans. We further characterized the PexRD12/31 family of RXLR-WY effectors, which associate and co-localize with components of the vesicle trafficking machinery. One member of this family, PexRD31, increased the number of FYVE positive vesicles in N. benthamiana cells. FYVE positive vesicles also accumulated in leaf cells near P. infestans hyphae, indicating that the pathogen may enhance endosomal trafficking during infection. We anticipate that the interactome dataset we generated will serve as a useful community resource for functional studies of P. infestans effectors and of effector-targeted host processes.

plant biology↗