Gopi K. Podila

Comparative Proteomic Analysis of Botrytis cinerea Secretome

Botrytis cinerea (B. cinerea) is a filamentous fungus infecting more than 200 plant species, causing significant economic losses worldwide. Secreted proteins are released as an initial response of the fungus to its plant host. We report the use of a high-throughput LC-MS/MS approach to analyze B. cinerea BO5.10 secreted proteins. Secretions were collected from fungus grown on a solid substrate of cellophane membrane while mock infecting media supplemented with the extract of full red tomato, ripened strawberry or Arabidopsis leaf extract. Overall, 89 B. cinerea proteins were identified from all growth conditions. Sixty proteins were predicted to contain a SignalP motif indicating the extracellular location of the proteins. Seven proteins were observed in all the growth conditions implying a constitutive nature of their secretion. Identified in the secretions were transport proteins, proteins well-characterized for carbohydrate metabolism, peptidases, oxidation/reduction, and pathogenicity factors that provide important insights into how B. cinerea may use secreted proteins for plant infection and colonization.

Using next generation transcriptome sequencing to predict an ectomycorrhizal metabolome

BackgroundMycorrhizae, symbiotic interactions between soil fungi and tree roots, are ubiquitous in terrestrial ecosystems. The fungi contribute phosphorous, nitrogen and mobilized nutrients from organic matter in the soil and in return the fungus receives photosynthetically-derived carbohydrates. This union of plant and fungal metabolisms is the mycorrhizal metabolome. Understanding this symbiotic relationship at a molecular level provides important contributions to the understanding of forest ecosystems and global carbon cycling.ResultsWe generated next generation short-read transcriptomic sequencing data from fully-formed ectomycorrhizae between Laccaria bicolor and aspen (Populus tremuloides) roots. The transcriptomic data was used to identify statistically significantly expressed gene models using a bootstrap-style approach, and these expressed genes were mapped to specific metabolic pathways. Integration of expressed genes that code for metabolic enzymes and the set of expressed membrane transporters generates a predictive model of the ectomycorrhizal metabolome. The generated model of mycorrhizal metabolome predicts that the specific compounds glycine, glutamate, and allantoin are synthesized by L. bicolor and that these compounds or their metabolites may be used for the benefit of aspen in exchange for the photosynthetically-derived sugars fructose and glucose.ConclusionsThe analysis illustrates an approach to generate testable biological hypotheses to investigate the complex molecular interactions that drive ectomycorrhizal symbiosis. These models are consistent with experimental environmental data and provide insight into the molecular exchange processes for organisms in this complex ecosystem. The method used here for predicting metabolomic models of mycorrhizal systems from deep RNA sequencing data can be generalized and is broadly applicable to transcriptomic data derived from complex systems.

High efficiency poplar transformation

With the completion of the poplar tree genome database, Populus species have become one of the most useful model systems for the study of woody plant biology. Populus tremuloides (quaking aspen) is the most wide-spread tree species in North America, and its rapid growth generates the most abundant wood-based biomass out of any other plant species. To study such beneficial traits, there is a need for easier and more efficient transformation procedures that will allow the study of large numbers of tree genes. We have developed transformation procedures that are suitable for high-throughput format transformations using either Agrobacterium tumefaciens to produce transformed trees or Agrobacterium rhizogenes to generate hairy roots. Our method uses Agrobacterium inoculated aspen seedling hypocotyls followed by direct thidiazuron (TDZ)-mediated shoot regeneration on selective media. Transformation was verified through β-glucuronidase (GUS) reporter gene expression in all tree tissues, PCR amplification of appropriate vector products from isolated genomic DNA, and northern hybridization of incorporated and expressed transgenes. The hairy root protocol follows the same inoculation procedures and was tested using GUS reporter gene integration and antibiotic selection. The benefit of these procedures is that they are simple and efficient, requiring no maintenance of starting materials and allowing fully formed transgenic trees (or hairy roots) to be generated in only 3–4xa0months, rather than the 6–12xa0months required by more traditional methods. Likewise, the fact that the protocols are amenable to high-throughput formats makes them better suited for large-scale functional genomics studies in poplars.

Transcriptomic comparison in the leaves of two aspen genotypes having similar carbon assimilation rates but different partitioning patterns under elevated [CO2].

This study compared the leaf transcription profiles, physiological characteristics and primary metabolites of two Populus tremuloides genotypes (clones 216 and 271) known to differ in their responses to long-term elevated [CO2] (e[CO2]) at the Aspen free-air CO2 enrichment site near Rhinelander, WI, USA. The physiological responses of these clones were similar in terms of photosynthesis, stomatal conductance and leaf area index under e[CO2], yet very different in terms of growth enhancement (0-10% in clone 216; 40-50% in clone 271). Although few genes responded to long-term exposure to e[CO2], the transcriptional activity of leaf e[CO2]-responsive genes was distinctly different between the clones, differentially impacting multiple pathways during both early and late growing seasons. An analysis of transcript abundance and carbon/nitrogen biochemistry suggested that the CO2-responsive clone (271) partitions carbon into pathways associated with active defense/response to stress, carbohydrate/starch biosynthesis and subsequent growth. The CO2-unresponsive clone (216) partitions carbon into pathways associated with passive defense (e.g. lignin, phenylpropanoid) and cell wall thickening. This study indicates that there is significant variation in expression patterns between different tree genotypes in response to long-term exposure to e[CO2]. Consequently, future efforts to improve productivity or other advantageous traits for carbon sequestration should include an examination of genetic variability in CO2 responsiveness.

Characterization of PTM5 in aspen trees: a MADS-box gene expressed during woody vascular development

The vascular component of trees possesses some of the most specialized processes active in the formation of roots, stems, and branches, and its wood component continues to be of primary importance to our daily lives. The molecular mechanisms of wood development, however, remain poorly understood with few well-characterized regulatory genes. We have identified a vascular tissue-specific MADS-box gene, Populus tremuloides MADS-box 5 (PTM5) that is expressed in differentiating primary and secondary xylem and phloem. Phylogenetic analysis has shown that PTM5 is a member of the SOC1/TM3 class of MADS-box genes. Temporal expression analysis of PTM5 in staged vascular cambium and other tissues indicated that PTM5 expression is seasonal and is limited to spring wood formation and rapidly expanding floral catkins. Spatial expression analysis using in situ hybridization revealed that PTM5 expression is localized within a few layers of differentiating vascular cambium and xylem tissues as well as the vascular bundles of expanding catkins. Since many MADS-box genes are known to act as transcription factors, these results suggest that the coordinated expression of PTM5 with other vascular developmental genes may be a hallmark of the complex events that lead to the formation of the woody plant body.

Using deep RNA sequencing for the structural annotation of the Laccaria bicolor mycorrhizal transcriptome.

Background Accurate structural annotation is important for prediction of function and required for in vitro approaches to characterize or validate the gene expression products. Despite significant efforts in the field, determination of the gene structure from genomic data alone is a challenging and inaccurate process. The ease of acquisition of transcriptomic sequence provides a direct route to identify expressed sequences and determine the correct gene structure. Methodology We developed methods to utilize RNA-seq data to correct errors in the structural annotation and extend the boundaries of current gene models using assembly approaches. The methods were validated with a transcriptomic data set derived from the fungus Laccaria bicolor, which develops a mycorrhizal symbiotic association with the roots of many tree species. Our analysis focused on the subset of 1501 gene models that are differentially expressed in the free living vs. mycorrhizal transcriptome and are expected to be important elements related to carbon metabolism, membrane permeability and transport, and intracellular signaling. Of the set of 1501 gene models, 1439 (96%) successfully generated modified gene models in which all error flags were successfully resolved and the sequences aligned to the genomic sequence. The remaining 4% (62 gene models) either had deviations from transcriptomic data that could not be spanned or generated sequence that did not align to genomic sequence. The outcome of this process is a set of high confidence gene models that can be reliably used for experimental characterization of protein function. Conclusions 69% of expressed mycorrhizal JGI “best” gene models deviated from the transcript sequence derived by this method. The transcriptomic sequence enabled correction of a majority of the structural inconsistencies and resulted in a set of validated models for 96% of the mycorrhizal genes. The method described here can be applied to improve gene structural annotation in other species, provided that there is a sequenced genome and a set of gene models.

cDNA and derived amino acid sequence of the chloroplastic copper/zinc-superoxide dismutase from aspen (Populus tremuloides)

aerobic and many anaerobic are Table 1. Characteristics of the chloroplastic Cu/Zn-SOD from aspen subiect to oxidative stress resulting from the deleterious efv fects of reduced oxygen species. SOD has been identified as a key enzyme in protecting the cells from oxidative damage by catalyzing the dismutation of superoxide radicals (O2-) to hydrogen peroxide and molecular oxygen (Fridovich, 1986). Organism:

Populus Rhizosphere and the Ectomycorrhizal Interactome

Interactions between trees such as Populus and their associated soil microbiome significantly impact the net plant productivity (NPP), carbon biosequestration and carbon allocation. In this chapter we discuss new insights learned from genomic-level studies involving Populus and its soil microbial community, especially symbiotic ectomycorrhizal interactions, focusing on the communication and signaling in the soil between the tree roots and the mycorrhizal fungi, effect of root exudates and fungal enzymes in the turn over and translocation of mineral nutrients, nitrogen and soil organic matter (SOM). Future prospects of functional genomic studies on plant and fungal components to improve carbon sequestration and partitioning are outlined. Studies of the Populus ectomycorrhizal interactome in the soil are critical to long-term ecosystem productivity and greatly impact planets atmosphere.

Cold induced Botrytis cinerea enolase (BcEnol-1) functions as a transcriptional regulator and is controlled by cAMP.

Botrytis cinerea is a necrotrophic fungal plant pathogen that can survive, grow and infect crops under cold stress. In an attempt to understand the molecular mechanisms leading to cold tolerance of this phytopathogen, we identified an enolase, BcEnol-1. BcEnol-1 encodes a 48xa0kDa protein that shows high identity to yeast, Arabidopsis and human enolases (72, 63 and 63%, respectively). Northern analysis confirms that an increase in transcript abundance of BcEnol-1 was observed when B. cinerea mycelium was shifted from 22 to 4°C. In order to understand its regulation during cold stress, BcEnol-1 expression was studied in B. cinerea mutants viz Δbcg1 (mutant of B. cinerea for bcg1), Δbcg3 (mutant of B. cinerea for bcg3) and Δbac (mutant of B. cinerea for adenylate cyclase). A decrease in enolase expression in these mutants was observed during cold stress suggesting enolase activation by a cAMP mediated cascade. Expression of enolase was restored with the exogenous addition of cAMP to the Δbac mutant. Recombinant enolase protein was also found to bind to the promoter elements of transcripts belonging to the Zinc-C6 protein family and calpain like proteases. Based on these results we conclude that enolase from Botrytis is cold responsive, influenced by cAMP and acts putatively as a transcriptional regulator.

Nucleotide Sequence of a Populus Tremuloides Gene Encoding Bispecific Caffeic Acid/5-Hydroxyferulic Acid O-Methyltransferase

Bi-specific OMT (EC 2.1.1.68) catalyzes the meta-specific methylation of caffeic acid and 5-hydroxyferulic acid to ferulic acid and sinapic acid, respectively (Higuchi, 1990). It is one of the key enzymes in angiosperm dicots mediating the formation of unique precursors for syringyl lignin, which is generally absent in gymnosperms (Higuchi, 1990). OMT has been purified and its cDNA clones isolated from Populus tremuloides (Bugos et al., 1991, 19921, Populus deltoides X Populus trichocarpa (Dumas et al., 1992), and severa1 other plants (Gowri et al., 1991; Callazo et al., 1992; Jaeck et al., 1992; Pellegrini et al., 1993). In this paper we report the nucleotide sequence encoding an OMT genomic clone, designated PTOMT, obtained from a n angiosperm tree species. Two oligonucleotide primers corresponding to nucleotide positions 65 to 82 and to positions 1370 to 1388 of a full-length OMT cDNA (Ptomtl) from secondary developing xylem tissue of P. tremuloides (Bugos et al., 1991) were used for PCR using P. tremuloides total cellular DNA as a template. A 2.7-kb DNA fragment was amplified, subcloned, and sequenced in its entirety in both directions. The P. tremuloides POMT gene contains four exons and three introns (Table I). downstream from the translation start codon. The amino acid sequence derived from this P. tremuloides genomic DNA is 99.7% homologous with that from Ptomtl (Bugos et al., 1991).