Category: Publications

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.

Cao FY, DeFalco TA, Moeder W, Li B, Gong Y, Liu XM, Taniguchi M, Lumba S, Toh S, Shan L, Ellis B, Desveaux D, Yoshioka K

BMC Plant Biol. 2018 Sep;18(1):211

PMID: 30261844

Abstract

BACKGROUND: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses.

RESULTS: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively.

CONCLUSIONS: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

Fitzpatrick CR, Lu-Irving P, Copeland J, Guttman DS, Wang PW, Baltrus DA, Dlugosch KM, Johnson MTJ

Microbiome 2018 Aug;6(1):144

PMID: 30121081

Abstract

BACKGROUND: The ability to efficiently characterize microbial communities from host individuals can be limited by co-amplification of host organellar sequences (mitochondrial and/or plastid), which share a common ancestor and thus sequence similarity with extant bacterial lineages. One promising approach is the use of sequence-specific peptide nucleic acid (PNA) clamps, which bind to, and block amplification of, host-derived DNA. Universal PNA clamps have been proposed to block host plant-derived mitochondrial (mPNA) and plastid (pPNA) sequences at the V4 16S rRNA locus, but their efficacy across a wide range of host plant species has not been experimentally tested.

RESULTS: Using the universal PNA clamps, we amplified and sequenced root microbial communities from replicate individuals of 32 plant species with a most recent common ancestor inferred at 140 MYA. We found the average rate of host plastid contamination across plant species was 23%, however, particular lineages exhibited much higher rates (62-94%), with the highest levels of contamination occurring in the Asteraceae. We investigated chloroplast sequence variation at the V4 locus across 500 land plant species (Embryophyta) and found six lineages with mismatches between plastid and the universal pPNA sequence, including all species within the Asteraceae. Using a modified pPNA for the Asteraceae sequence, we found (1) host contamination in Asteraceae species was reduced from 65 to 23%; and (2) host contamination in non-Asteraceae species was increased from 12 to 69%. These results demonstrate that even single nucleotide mismatches can lead to drastic reductions in pPNA efficacy in blocking host amplification. Importantly, we found that pPNA type (universal or modified) had no effect on the detection of individual bacterial taxa, or estimates of within and between sample bacterial diversity, suggesting that our modification did not introduce bias against particular bacterial lineages.

CONCLUSIONS: When high similarity exists between host organellar DNA and PCR target sequences, PNA clamps are an important molecular tool to reduce host contamination during amplification. Here, we provide a validated framework to modify universal PNA clamps to accommodate host variation in organellar sequences.

Sheppard SK, Guttman DS, Fitzgerald JR

Nat. Rev. Genet. 2018 Jul;

PMID: 29973680

Abstract

Some bacteria can transfer to new host species, and this poses a risk to human health. Indeed, an estimated 60% of all human pathogens have originated from other animal species. Similarly, human-to-animal transitions are recognized as a major threat to sustainable livestock production, and emerging pathogens impose an increasing burden on crop yield and global food security. Recent advances in high-throughput sequencing technologies have enabled comparative genomic analyses of bacterial populations from multiple hosts. Such studies are providing new insights into the evolutionary processes that underpin the establishment of bacteria in new host niches. A better understanding of the genetic and mechanistic basis for bacterial host adaptation may reveal novel targets for controlling infection or inform the design of approaches to limit the emergence of new pathogens.

Gritsunov A, Peek J, Diaz Caballero J, Guttman D, Christendat D

Plant J. 2018 Jun;

PMID: 29890023

Abstract

Quinate is produced and used by many plants in the biosynthesis of chlorogenic acids (CGA). CGA are astringent which serves as deterrents of herbivory. They also function as antifungal agents and have potent antioxidant properties. Quinate is produced at a branch point of shikimate biosynthesis by the enzyme, quinate dehydrogenase (QDH). However, little information exists on the identity and biochemical properties of plant QDHs. In this study, we utilized structural and bioinformatics approaches to establish a QDH-specific primary sequence motif. Using this motif, we identified QDHs from diverse plants and confirmed their activity by recombinant protein production and kinetic assays. Through a detailed phylogenetic analysis, we show that plant QDHs arose directly from bifunctional dehydroquinate dehydratase-shikimate dehydrogenases (DHQD-SDHs) through different convergent evolutionary events. Illustrated by our findings that eudicots and conifers QDHs arose early in vascular plant evolution whereas Brassicaceae QDHs emerged later. This process of QDH recurrent evolution is further demonstrated by the fact that this family of proteins evolved NAD and NADP specificity independently in eudicots. The acquisition of QDH activity by these proteins was accompanied by the inactivation or functional evolution of the DHQD domain, as verified by enzyme activity assays and as reflected in the loss of key DHQD active site residues. The implications of QDH activity and evolution are discussed in terms of plant growth and development. This article is protected by copyright. All rights reserved.

Zhang HM, Xia HL, Jiang HR, Mao YJ, Qu KX, Huang BZ, Gong YC, Yang ZP

Genome 2018 Jun;

PMID: 29883552

Abstract

Intramuscular fat (IMF) content and fatty acid (FA) composition vary significantly across beef cattle breeds, which play important roles in taste and nutritional value. However, the molecular mechanisms underlying these phenotypic differences remain unknown. The present study compared meat quality traits between Yunling cattle and Chinese Simmental cattle. Yunling cattle showed a lower IMF content and proportion of monounsaturated fatty acids (MUFA), as well as higher proportions of saturated fatty acids (SFA), polyunsaturated fatty acids (PUFA) and short chain fatty acids (sc-FA) in the longissimus dorsi (LD) muscle than Chinese Simmental cattle. To further identify the candidate genes and pathways responsible for these phenotypic differences, the transcriptome of LD muscle from the two breeds were measured using RNA-seq. A total of 1,347 differentially expressed genes were identified. The major metabolic pathways that were differentially modulated were lipolysis and glycometabolism. Yunling cattle showed a higher expression of lipolysis genes (ALDH9A1, ACSL5, ACADM, ACAT2, ACOT2) and a lower expression of genes related to glycometabolism (PGM1, GALM, PGM1, GPI, LDHA). This research identified candidate genes and pathways for IMF content and FA composition in the LD muscle of beef cattle, which may facilitate the design of new selection strategies to improve meat quality.

Fischer C, DeFalco TA, Karia P, Snedden WA, Moeder W, Yoshioka K, Dietrich P

Plant Cell Physiol. 2017 Jul;58(7):1208-1221

PMID: 28419310

Abstract

Ca2+ serves as a universal second messenger in eukaryotic signaling pathways, and the spatial and temporal patterns of Ca2+ concentration changes are determined by feedback and feed-forward regulation of the involved transport proteins. Cyclic nucleotide-gated channels (CNGCs) are Ca2+-permeable channels that interact with the ubiquitous Ca2+ sensor calmodulin (CaM). CNGCs interact with CaMs via diverse CaM-binding sites, including an IQ-motif, which has been identified in the C-termini of CNGC20 and CNGC12. Here we present a family-wide analysis of the IQ-motif from all 20 Arabidopsis CNGC isoforms. While most of their IQ-peptides interacted with conserved CaMs in yeast, some were unable to do so, despite high sequence conservation across the family. We showed that the CaM binding ability of the IQ-motif is highly dependent on its proximal and distal vicinity. We determined that two alanine residues positioned N-terminal to the core IQ-sequence play a significant role in CaM binding, and identified a polymorphism at this site that promoted or inhibited CaM binding in yeast. Through detailed biophysical analysis of the CNGC2 IQ-motif, we found that this polymorphism specifically affected the Ca2+-independent interactions with the C-lobe of CaM. This same polymorphism partially suppressed the induction of programmed cell death by CNGC11/12 in planta. Our work expands the model of CNGC regulation, and posits that the C-lobe of apo-CaM is permanently associated with the channel at the N-terminal part of the IQ-domain. This mode allows CaM to function as a Ca2+-sensing regulatory subunit of the channel complex, providing a mechanism by which Ca2+ signals may be fine-tuned.

DeFalco TA, Toyota M, Phan V, Karia P, Moeder W, Gilroy S, Yoshioka K

Plant Cell Physiol. 2017 Jul;58(7):1173-1184

PMID: 28482045

Abstract

Ca2+ signaling is a central component of plant biology; however, direct analysis of in vivo Ca2+ levels is experimentally challenging. In recent years, the use of genetically encoded Ca2+ indicators has revolutionized the study of plant Ca2+ signaling, although such studies have been largely restricted to the model plant Arabidopsis. We have developed stable transgenic Nicotiana benthamiana and Nicotiana tabacum lines expressing the single-wavelength fluorescent Ca2+ indicator, GCaMP3. Ca2+ levels in these plants can be imaged in situ using fluorescence microscopy, and these plants can be used qualitatively and semi-quantitatively to evaluate Ca2+ signals in response to a broad array of abiotic or biotic stimuli, such as cold shock or pathogen-associated molecular patterns (PAMPs). Furthermore, these tools can be used in conjunction with well-established N. benthamiana techniques such as virus-induced gene silencing (VIGS) or transient heterologous expression to assay the effects of loss or gain of function on Ca2+ signaling, an approach which we validated via silencing or transient expression of the PAMP receptors FLS2 (Flagellin Sensing 2) or EFR (EF-Tu receptor), respectively. Using these techniques, along with chemical inhibitor treatments, we demonstrate how these plants can be used to elucidate the molecular components governing Ca2+ signaling in response to specific stimuli.