Author: biota

Yu X, Xu G, Li B, de Souza Vespoli L, Liu H, Moeder W, Chen S, de Oliveira MVV, Ariádina de Souza S, Shao W, Rodrigues B, Ma Y, Chhajed S, Xue S, Berkowitz GA, Yoshioka K, He P, Shan L.

Curr Biol. 2019 Oct 23. pii: S0960-9822(19)31178-9. doi: 10.1016/j.cub.2019.09.018.

PMID: 31679931

Abstract:

Cell death is a vital and ubiquitous process that is tightly controlled in all organisms. However, the mechanisms underlying precise cell death control remain fragmented. As an important shared module in plant growth, development, and immunity, Arabidopsis thaliana BRASSINOSTEROID INSENSITIVE 1-associated receptor kinase 1 (BAK1) and somatic embryogenesis receptor kinase 4 (SERK4) redundantly and negatively regulate plant cell death. By deploying an RNAi-based genetic screen for bak1/serk4 cell death suppressors, we revealed that cyclic nucleotide-gated channel 20 (CNGC20) functions as a hyperpolarization-activated Ca²⁺-permeable channel specifically regulating bak1/serk4 cell death. BAK1 directly interacts with and phosphorylates CNGC20 at specific sites in the C-terminal cytosolic domain, which in turn regulates CNGC20 stability. CNGC19, the closest homolog of CNGC20 with a low abundance compared with CNGC20, makes a quantitative genetic contribution to bak1/serk4 cell death only in the absence of CNGC20, supporting the biochemical data showing homo- and heteromeric assembly of the CNGC20 and CNGC19 channel complexes. Transcripts of CNGC20 and CNGC19 are elevated in bak1/serk4 compared with wild-type plants, further substantiating a critical role of homeostasis of CNGC20 and CNGC19 in cell death control. Our studies not only uncover a unique regulation of ion channel stability by cell-surface-resident receptor kinase-mediated phosphorylation but also provide evidence for fine-tuning Ca²⁺ channel functions in maintaining cellular homeostasis by the formation of homo- and heterotetrameric complexes.

Robertson SJ, Lemire P, Maughan H, Goethel A, Turpin W, Bedrani L, Guttman DS, Croitoru K, Girardin SE, Philpott DJ

Cell Rep. 2019 May 7;27(6):1910-1919.e2. doi: 10.1016/j.celrep.2019.04.023

PMID: 31067473

Abstract:

The intestinal microbiota is a fundamental factor that broadly influences physiology. Thus, studies using transgenic animals should be designed to limit the confounding effects of microbiota variation between strains. Here, we report the impact on intestinal microbiota of co-housed versus F2-generation littermates, two commonly used techniques to standardize microbiota in animal models. Our results establish that while fecal microbiota is partially normalized by extended co-housing, mucosal communities associated with the proximal colon and terminal ileum remain stable and distinct. In contrast, strain inter-crossing to generate F2 littermates allows robust microbiota standardization in fecal, colon, and ileum sampling locations. Using reciprocal inter-crosses of P1 parents, we identify dissymmetry in F2 community structures caused by maternal transmission, in particular of the Verrucomicrobiaceae. Thus, F2 littermate animals from a unidirectional P1 cross should be used as a standard method to minimize the influence of the microbiota in genotype-phenotype studies.

Dillon MM, Almeida RND, Laflamme B, Martel A, Weir BS, Desveaux D, Guttman DS

Front Plant Sci. 2019 Apr 5;10:418. doi: 10.3389/fpls.2019.00418

PMID: 31024592

Abstract

Diverse Gram-negative pathogens like Pseudomonas syringae employ type III secreted effector (T3SE) proteins as primary virulence factors that combat host immunity and promote disease. T3SEs can also be recognized by plant hosts and activate an effector triggered immune (ETI) response that shifts the interaction back toward plant immunity. Consequently, T3SEs are pivotal in determining the virulence potential of individual P. syringae strains, and ultimately help to restrict P. syringae pathogens to a subset of potential hosts that are unable to recognize their repertoires of T3SEs. While a number of effector families are known to be present in the P. syringaespecies complex, one of the most persistent challenges has been documenting the complex variation in T3SE contents across a diverse collection of strains. Using the entire pan-genome of 494 P. syringae strains isolated from more than 100 hosts, we conducted a global analysis of all known and putative T3SEs. We identified a total of 14,613 putative T3SEs, 4,636 of which were unique at the amino acid level, and show that T3SE repertoires of different P. syringae strains vary dramatically, even among strains isolated from the same hosts. We also find substantial diversification within many T3SE families, and in many cases find strong signatures of positive selection. Furthermore, we identify multiple gene gain and loss events for several families, demonstrating an important role of horizontal gene transfer (HGT) in the evolution of P. syringae T3SEs. These analyses provide insight into the evolutionary history of P. syringae T3SEs as they co-evolve with the host immune system, and dramatically expand the database of P. syringae T3SEs alleles.

Cao FY, Khan M, Taniguchi M, Mirmiran A, Moeder W, Lumba S, Yoshioka K, Desveaux D

Plant J. 2019 May 31. doi: 10.1111/tpj.14425

PMID: 31148337

Abstract

The phytopathogen Pseudomonas syringae delivers into host cells type III secreted effectors (T3SEs) that promote virulence. One virulence mechanism employed by T3SEs is to target hormone signalling pathways to perturb hormone homeostasis. The phytohormone abscisic acid (ABA) influences interactions between various phytopathogens and their plant hosts, and has been shown to be a target of P. syringae T3SEs. In order to provide insight into how T3SEs manipulate ABA responses, we generated an ABA-T3SE interactome network (ATIN) between P. syringae T3SEs and Arabidopsis proteins encoded by ABA-regulated genes. ATIN consists of 476 yeast two-hybrid interactions between 97 Arabidopsis ABA-regulated proteins and 56 T3SEs from four pathovars of P. syringae. We demonstrate that T3SE interacting proteins are significantly enriched for proteins associated with transcription. In particular, the ETHYLENE RESPONSIVE FACTOR (ERF) family of transcription factors are highly represented. We show that ERF105 and ERF8 displayed a role in defense against P. syringae, supporting our overall observation that T3SEs of ATIN converge on proteins that influence plant immunity. In addition, we demonstrate that T3SEs that interact with a large number of ABA-regulated proteins can influence ABA responses. One of these T3SEs, HopF3_Pph6 , inhibits the function of ERF8, which influences both ABA-responses and plant immunity. These results provide a potential mechanism for how HopF3_Pph6 manipulates ABA-responses to promote P. syringae virulence, and also demonstrate the utility of ATIN as a resource to study the ABA-T3SE interface.

Mott GA, Smakowska-Luzan E, Pasha A, Parys K, Howton TC, Neuhold J, Lehner A, Grünwald K, Stolt-Bergner P, Provart NJ, Mukhtar MS, Desveaux D, Guttman DS, Belkhadir Y.

Sci Data. 2019 Feb 26;6:190025.

PMID: 30806640

Abstract

Plants use surface receptors to perceive information about many aspects of their local environment. These receptors physically interact to form both steady state and signalling competent complexes. The signalling events downstream of receptor activation impact both plant developmental and immune responses. Here, we present a comprehensive study of the physical interactions between the extracellular domains of leucine-rich repeat receptor kinases (LRR-RKs) in Arabidopsis. Using a sensitized assay, we tested reciprocal interactions among 200 of the 225 Arabidopsis LRR-RKs for a total search space of 40,000 interactions. Applying a stringent statistical cut-off and requiring that interactions performed well in both bait-prey and prey-bait orientations resulted in a high-confidence set of 567 bidirectional interactions. Additionally, we identified a total of 2,586 unidirectional interactions, which passed our stringent statistical cut-off in only one orientation. These datasets will guide further investigation into the regulatory roles of LRR-RKs in plant developmental and immune signalling decisions.

Moeder W, Phan V, Yoshioka K

Plant Sci. 2019 Feb;279:19-26

PMID: 30709488

Abstract

Ca is a universal second messenger in many signaling pathways in all eukaryotes including plants. Transient changes in [Ca]cyt are rapidly generated upon a diverse range of stimuli such as drought, heat, wounding, and biotic stresses (infection by pathogenic and symbiotic microorganisms), as well as developmental cues. It has been known for a while that [Ca]cyt transient signals play crucial roles to activate plant immunity and recently significant progresses have been made in this research field. However the identity and regulation of ion channels that are involved in defense related Ca signals are still enigmatic. Members of two ligand gated ion channel families, glutamate receptor-like channels (GLRs) and cyclic nucleotide-gated channels (CNGCs) have been implicated in immune responses; nevertheless more precise data to understand their direct involvement in the creation of Ca signals during immune responses is necessary. Furthermore, the study of other ion channel groups is also required to understand the whole picture of the intra- and inter-cellular Ca signalling network. In this review we summarize Ca signals in plant immunity from an ion channel point of view and discuss future challenges in this exciting research field.

Clark ST, Sinha U, Zhang Y, Wang PW, Donaldson SL, Coburn B, Waters VJ, Yau YCW, Tullis DE, Guttman DS, Hwang DM

Int. J. Antimicrob. Agents 2019 Jan;

PMID: 30664925

Abstract

Determining the mechanisms that modulate β-lactam resistance in clinical P. aeruginosa isolates can be challenging, as the molecular profiles identified in mutation- or expression-based resistance determinant screens may not correlate with in vitro phenotypes. One of the lesser studied resistance mechanisms in P. aeruginosa is the modification of penicillin binding protein 3 (pbpB/ftsI). Here, we report that nonsynonymous polymorphisms within pbpB frequently occur among β-lactam resistant sputum isolates, and are associated with unique antibiotic susceptibility patterns. Longitudinally collected isolates (n=126) from cystic fibrosis (CF) patients with or without recent β-lactam therapy or of non-clinical origin were tested for susceptibility to six β-lactams (aztreonam, ceftazidime, cefsulodin, cefepime, meropenem and piperacillin). Known β-lactam resistance mechanisms were characterized by PCR-based methods, and polymorphisms in the transpeptidase-encoding domain of pbpB identified by sequencing. Twelve nonsynonymous polymorphisms were detected among 86 isolates (67%) from five CF patients with a history of β-lactam therapy, compared with only one polymorphism in 30 (3.3%) from three patients who had not received β-lactam treatments. No nonsynonymous polymorphisms were found in ten environmental isolates. Multiple pbpB alleles, often with different combinations of polymorphisms, were detected within the population of strains from each CF patient for up to 2.6 years. Traditional patterns of ampC or mexA de-repression, reduced expression of oprD or the presence of extended spectrum β-lactamases were not observed in resistant isolates with nonsynonymous polymorphisms in pbpB. Our findings suggest that pbpB is a common adaptive target, and may contribute to the development of β-lactam resistance in P. aeruginosa.

Diaz Caballero J, Clark ST, Wang PW, Donaldson SL, Coburn B, Tullis DE, Yau YCW, Waters VJ, Hwang DM, Guttman DS

PLoS Pathog. 2018 Dec;14(12):e1007453

PMID: 30532201

Abstract

Cystic fibrosis (CF) lung infections caused by members of the Burkholderia cepacia complex, such as Burkholderia multivorans, are associated with high rates of mortality and morbidity. We performed a population genomics study of 111 B. multivorans sputum isolates from one CF patient through three stages of infection including an early incident isolate, deep sampling of a one-year period of chronic infection occurring weeks before a lung transplant, and deep sampling of a post-transplant infection. We reconstructed the evolutionary history of the population and used a lineage-controlled genome-wide association study (GWAS) approach to identify genetic variants associated with antibiotic resistance. We found the incident isolate was basally related to the rest of the strains and more susceptible to antibiotics from three classes (β-lactams, aminoglycosides, quinolones). The chronic infection isolates diversified into multiple, distinct genetic lineages and showed reduced antimicrobial susceptibility to the same antibiotics. The post-transplant reinfection isolates derived from the same source as the incident isolate and were genetically distinct from the chronic isolates. They also had a level of susceptibility in between that of the incident and chronic isolates. We identified numerous examples of potential parallel pathoadaptation, in which multiple mutations were found in the same locus or even codon. The set of parallel pathoadaptive loci was enriched for functions associated with virulence and resistance. Our GWAS analysis identified statistical associations between a polymorphism in the ampD locus with resistance to β-lactams, and polymorphisms in an araC transcriptional regulator and an outer membrane porin with resistance to both aminoglycosides and quinolones. Additionally, these three loci were independently mutated four, three and two times, respectively, providing further support for parallel pathoadaptation. Finally, we identified a minimum of 14 recombination events, and observed that loci carrying putative parallel pathoadaptations and polymorphisms statistically associated with β-lactam resistance were over-represented in these recombinogenic regions.

Huei-Yi Lee A, Bastedo DP, Youn JY, Lo T, Middleton MA, Kireeva I, Lee JY, Sharifpoor S, Baryshnikova A, Zhang J, Wang PW, Peisajovich SG, Costanzo M, Andrews BJ, Boone CM, Desveaux D, Guttman DS

G3 (Bethesda) 2018 Dec;

PMID: 30573466

Abstract

Gram-negative bacterial pathogens inject type III secreted effectors (T3SEs) directly into host cells to promote pathogen fitness by manipulating host cellular processes. Despite their crucial role in promoting virulence, relatively few T3SEs have well-characterized enzymatic activities or host targets. This is in part due to functional redundancy within pathogen T3SE repertoires as well as the promiscuity of individual T3SEs that can have multiple host targets. To overcome these challenges, we generated and characterized a collection of yeast strains stably expressing 75 T3SE constructs from the plant pathogen This collection is devised to facilitate heterologous genetic screens in yeast, a non-host organism, to identify T3SEs that target conserved eukaryotic processes. Among 75 T3SEs tested, we identified 16 that inhibited yeast growth on rich media and eight that inhibited growth on stress-inducing media. We utilized Pathogenic Genetic Array (PGA) screens to identify potential host targets of T3SEs. We focused on the acetyltransferase, HopZ1a, which interacts with plant tubulin and alters microtubule networks. To uncover putative HopZ1a host targets, we identified yeast genes with genetic interaction profiles most similar (i.e. congruent) to the PGA profile of HopZ1a and performed a functional enrichment analysis of these HopZ1a-congruent genes. We compared the congruence analyses above to previously described HopZ physical interaction datasets and identified kinesins as potential HopZ1a targets. Finally, we demonstrated that HopZ1a can target kinesins by acetylating the plant kinesins HINKEL and MKRP1, illustrating the utility of our T3SE-expressing yeast library to characterize T3SE functions.

Dillon MM, Thakur S, Almeida RND, Wang PW, Weir BS, Guttman DS

Genome Biol. 2019 Jan;20(1):3

PMID: 30606234

Abstract

BACKGROUND: Pseudomonas syringae is a highly diverse bacterial species complex capable of causing a wide range of serious diseases on numerous agronomically important crops. We examine the evolutionary relationships of 391 agricultural and environmental strains using whole-genome sequencing and evolutionary genomic analyses.

RESULTS: We describe the phylogenetic distribution of all 77,728 orthologous gene families in the pan-genome, reconstruct the core genome phylogeny using the 2410 core genes, hierarchically cluster the accessory genome, identify the diversity and distribution of type III secretion systems and their effectors, predict ecologically and evolutionary relevant loci, and establish the molecular evolutionary processes operating on gene families. Phylogenetic and recombination analyses reveals that the species complex is subdivided into primary and secondary phylogroups, with the former primarily comprised of agricultural isolates, including all of the well-studied P. syringae strains. In contrast, the secondary phylogroups include numerous environmental isolates. These phylogroups also have levels of genetic diversity typically found among distinct species. An analysis of rates of recombination within and between phylogroups revealed a higher rate of recombination within primary phylogroups than between primary and secondary phylogroups. We also find that “ecologically significant” virulence-associated loci and “evolutionarily significant” loci under positive selection are over-represented among loci that undergo inter-phylogroup genetic exchange.

CONCLUSIONS: While inter-phylogroup recombination occurs relatively rarely, it is an important force maintaining the genetic cohesion of the species complex, particularly among primary phylogroup strains. This level of genetic cohesion, and the shared plant-associated niche, argues for considering the primary phylogroups as a single biological species.