A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays. Here, we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants. We developed vectors that enable Tn7 transposon-mediated integration of the luxCDABE luciferase operon into a specific genomic location found ubiquitously across bacterial phyla. These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas, Rhizobium (Agrobacterium), and Ralstonia. Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv. tomato DC3000 (Pto-lux) reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana, Solanum lycopersicum, Nicotiana benthamiana, and Marchantia polymorpha. We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains. Importantly, quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth, with a dynamic range of four orders of magnitude. Moreover, macroscopic bioluminescence imaging illuminated the spatial patterns of Pto-lux growth in/on inoculated plant tissues. In conclusion, our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions

Isolation and characterization of the E2F-like gene in plants11The sequences reported in this paper have been deposited in the GenBank database (accession numbers AB025347 for NtE2F and AB025029 for tobacco PCNA).

AbstractThe transcription factor E2F regulates the expression of genes involved in the progression of G1/S transition and DNA replication in mammalian cells. We cloned and characterized a cDNA (NtE2F) corresponding to a E2F homolog of tobacco (Nicotiana tabacum). The transcription of NtE2F was induced as cells progressed from G1 to the S phase and expressed much earlier than that of the proliferating cell nuclear antigen (PCNA) gene. We demonstrated that NtE2F can interact with the tobacco retinoblastoma (Rb)-related protein in a yeast two-hybrid assay. To further characterize NtE2F, the trans-activation activity of NtE2F was examined by using a transient assay in the tobacco Bright Yellow-2 (BY-2) cells with NtE2F fused to the DNA-binding domain of the yeast transcriptional activator GAL4. NtE2F activated the transcription of the β-glucuronidase (GUS) reporter gene driven by a cauliflower mosaic virus (CaMV) 35S core promoter containing the GAL4-binding sequence. This is the first report of the identification of a functionally equivalent E2F-like gene in plants

Algal protein kinase, Triacylglycerol Accumulation Regulator 1, modulates cell viability and gametogenesis in carbon/nitrogen imbalanced conditions

Nutrient-deprived microalgae accumulate triacylglycerol (TAG) in lipid droplets. A dual-specificity tyrosine phosphorylation-regulated kinase, TAG accumulation regulator 1 (TAR1) has been shown to be required for acetate-dependent TAG accumulation and the degradation of chlorophyll and photosynthesis-related proteins in photomixotrophic nitrogen (N)-deficient conditions (Kajikawa et al. 2015). However, this previous report only examined particular condition. Here, we report that in photoautotrophic N-deficient conditions, tar1-1 cells, with a mutation in the TAR1 gene, maintained higher levels of cell viability and lower levels of hydrogen peroxide generation and accumulated higher levels of TAG and starch compared with those of wild-type (WT) cells with bubbling of air containing 5% carbon dioxide. Transcriptomic analyses suggested that genes involved in the scavenging of reactive oxygen species are not repressed in tar1-1 cells. In contrast, the mating efficiency and mRNA levels of key regulatory genes for gametogenesis, MID, MTD, and FUS, were suppressed in tar1-1 cells. Among the TAR1-dependent phosphopeptides deduced by phosphoproteomic analysis, protein kinases and enzymes related to N assimilation and carbon (C) metabolism are of particular interest. Characterization of these putative downstream factors may elucidate the molecular pathway whereby TAR1 mediates cellular propagation and C and N metabolism in C/N-imbalanced stress conditions

Conserved Biochemical Defenses Underpin Host Responses to Oomycete Infection in an Early-Divergent Land Plant Lineage.

The expansion of plants onto land necessitated the evolution of robust defense strategies to protect against a wide array of microbial invaders. Whereas host responses to microbial colonization are extensively explored in evolutionarily young land plant lineages such as angiosperms, we know relatively little about plant-pathogen interactions in early-diverging land plants thought to better represent the ancestral state. Here, we define the transcriptional and proteomic response of the early-divergent liverwort Marchantia polymorpha to infection with the oomycete pathogen Phytophthora palmivora. We uncover a robust molecular response to oomycete colonization in Marchantia that consists of conserved land plant gene families. Direct macroevolutionary comparisons of host infection responses in Marchantia and the model angiosperm Nicotiana benthamiana further reveal a shared set of orthologous microbe-responsive genes that include members of the phenylpropanoid metabolic pathway. In addition, we identify a role for the Marchantia R2R3-MYB transcription factor MpMyb14 in activating phenylpropanoid (flavonoid) biosynthesis during oomycete infection. Mpmyb14 mutants infected with P. palmivora fail to activate phenylpropanoid biosynthesis gene expression and display enhanced disease susceptibility compared to wild-type plants. Conversely, the ectopic induction of MpMyb14 led to the accumulation of anthocyanin-like pigments and dramatically enhanced liverwort resistance to P. palmivora infection. Collectively, our results demonstrate that the Marchantia response to oomycete infection displays evolutionarily conserved features indicative of an ancestral pathogen deterrence strategy centered on phenylpropanoid-mediated biochemical defenses.Gatsby Charitable Foundation (GAT3395/GLD) Royal Society (UF160413, RGF\EA\180002) BBSRC OpenPlant initiative (BB/L014130/1) Natural Environment Research Council (NERC; NE/N00941X/1), Natural Sciences and Engineering Research Council of Canada (NSERC) postdoctoral fellowship, Max-Planck-Gesellschaft

A class III peroxidase PRX34 is a component of disease resistance in Arabidopsis

PRX34 mediates the oxidative burst in Arabidopsis. Here we characterized two additional Arabidopsis prx34 null mutants (prx34-2, prx34-3), besides the well-studied prx34-1. Due to a decrease in corresponding peroxidase, the activity that generates reactive oxygen species (ROS) was significantly lower in cell wall extracts of prx34-2 and prx34-3 plants. Consistently, the prx34-2 and prx34-3 exhibited reduced accumulation both of ROS and callose in Flg22-elicitor-treated leaves, leading to enhanced susceptibility to bacterial and fungal pathogens. In contrast, ectopic expression of PRX34 in the wild type caused enhanced resistance. PRX34 is thus a component for disease resistance in Arabidopsis

Gene modification by fast-track recombineering for cellular localization and isolation of components of plant protein complexes.

To accelerate the isolation of plant protein complexes and study cellular localization and interaction of their components, an improved recombineering protocol is described for simple and fast site-directed modification of plant genes in bacterial artificial chromosomes (BACs). Coding sequences of fluorescent and affinity tags were inserted into genes and transferred together with flanking genomic sequences of desired size by recombination into Agrobacterium plant transformation vectors using three steps of E. coli transformation with PCR-amplified DNA fragments. Application of fast-track recombineering is illustrated by the simultaneous labelling of CYCLINDEPENDENT KINASE D (CDKD) and CYCLIN H (CYCH) subunits of kinase module of TFIIH general transcription factor and the CDKD-activating CDKF;1 kinase with green fluorescent protein (GFP) and mCherry (green and red fluorescent protein) tags, and a PIPL (His18-StrepII-HA) epitope. Functionality of modified CDKF;1 gene constructs is verified by complementation of corresponding T-DNA insertion mutation. Interaction of CYCH with all three known CDKD homologues is confirmed by their co-localization and co-immunoprecipitation. Affinity purification and mass spectrometry analyses of CDKD;2, CYCH, and DNA-replication-coupled HISTONE H3.1 validate their association with conserved TFIIH subunits and components of CHROMATIN ASSEMBLY FACTOR 1, respectively. The results document that simple modification of plant gene products with suitable tags by fast-track recombineering is well suited to promote a wide range of protein interaction and proteomics studies

The leucine-rich repeat receptor kinase QSK1 is a novel regulator of PRR-RBOHD complex and is employed by the bacterial effector HopF2Pto_{Pto} to modulate plant immunity

Plants detect pathogens using cell-surface pattern recognition receptors (PRRs) like EFR and FLS2, which recognize bacterial EF-Tu and flagellin, respectively. These PRRs, belonging to the leucine-rich repeat receptor kinase (LRR-RK) family, activate the production of reactive oxygen species via the NADPH oxidase RBOHD. The PRR-RBOHD complex is tightly regulated to prevent unwarranted or exaggerated immune responses. However, certain pathogenic effectors can subvert these regulatory mechanisms, thereby suppressing plant immunity. To elucidate the intricate dynamics of the PRR-RBOHD complex, we conducted a comparative co-immunoprecipitation analysis using EFR, FLS2, and RBOHD. We identified QSK1, an LRR-RK, as a novel component of the PRR-RBOHD complex. QSK1 functions as a negative regulator of PRR-triggered immunity (PTI) by downregulating the abundance of FLS2 and EFR. QSK1 is targeted by the bacterial effector HopF2$_{Pto}$, a mono-ADP ribosyltransferase, resulting in the reduction of FLS2 and EFR levels through both transcriptional and transcription-independent pathways, thereby inhibiting PTI. Furthermore, HopF2$_{Pto}$ reduces transcript levels of PROSCOOP genes encoding important stress-regulated phytocytokines and their receptor MIK2. Importantly, HopF2Pto requires QSK1 for its accumulation and virulence functions within plants. In summary, our results provide novel insights into the mechanism by which HopF2$_{Pto}$ employs QSK1 to desensitize plants to pathogen attack. One Sentence Summary: QSK1, a novel component in the plant immune receptor complex, downregulates these receptors and phytocytokines, and is exploited by bacterial effector HopF2$_{Pto}$ to desensitize plants to pathogen attack

Variation in plant Toll/Interleukin-1 receptor domain protein dependence on ENHANCED DISEASE SUSCEPTIBILITY 1

Toll/Interleukin-1 receptor (TIR) domains are integral to immune systems across all kingdoms. In plants, TIRs are present in nucleotide-binding leucine-rich repeat (NLR) immune receptors, NLR-like, and TIR-only proteins. Although TIR-NLR and TIR signaling in plants require the ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) protein family, TIRs persist in species that have no EDS1 members. To assess whether particular TIR groups evolved with EDS1, we searched for TIR-EDS1 co-occurrence patterns. Using a large-scale phylogenetic analysis of TIR domains from 39 algal and land plant species, we identified 4 TIR families that are shared by several plant orders. One group occurred in TIR-NLRs of eudicots and another in TIR-NLRs across eudicots and magnoliids. Two further groups were more widespread. A conserved TIR-only group co-occurred with EDS1 and members of this group elicit EDS1-dependent cell death. In contrast, a maize (Zea mays) representative of TIR proteins with tetratricopeptide repeats was also present in species without EDS1 and induced EDS1-independent cell death. Our data provide a phylogeny-based plant TIR classification and identify TIRs that appear to have evolved with and are dependent on EDS1, while others have EDS1-independent activity