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Selected Competing Graduate Students

Caitlin Cridland, Department of Biochemistry  |  Inositol Pyrophosphates and the Cross Talk Between Lipids and Phosphate Sensing 
Phosphate (Pi) is an essential nutrient for plants, required for plant growth and seed viability. Under Pi stress, plants undergo dynamic morphological and metabolism changes to leverage available Pi, including the breakdown of membrane phospholipids. Plants have been shown to ‚Äúremodel‚Äù their lipid membrane profiles under phosphate starvation, degrading phospholipids in the cell membranes and utilizing the generated phosphorus for essential biological processes. By concomitantly inducing a phospholipid hydrolysis pathway and galactolipid biosynthetic pathway, membrane phospholipids are replaced by non-phosphorus containing galactolipids and sulfolipids. The inositol phosphate signaling pathway is a crucial element of the plant‚Äôs ability to respond to changing energy conditions.  Inositol phosphates (InsPs) are synthesized from the cyclic 6-carbon polyol scaffold, myo-inositol. Inositol hexakisphosphate (InsP6) is the most abundant InsP signalling molecule and can be phosphorylated further by VIP kinases, resulting in inositol pyrophosphates (PPx-InsPs). PPx-InsPs have high energy bonds, and have been linked to maintaining phosphate (Pi) and energy homeostasis in yeast. Using liquid chromatography-mass spectrometry and tandem mass spectrometry, we are examining the lipid profile of an Arabidopsis vip double mutant, in response to phosphate depletion, to address the role of PPx-InsPs in Pi sensing. Our results suggest that PPx-InsPs play a crucial role in Pi sensing and are involved in the regulation of lipid biosynthesis. 

 

Marco E Mechan, School of Plant and Environmental SciencesThe rain microbiome: A source of the plant leaf microbiome?
Plants live in association with a large diversity of microbial communities known as the plant microbiome. Root-associated bacteria have been found to be recruited from the surrounding soil; however, the source of the leaf microbiome has not been determined with confidence. Here we test the hypothesis that rain is a contributor to the plant leaf microbiome. We used 16S rRNA sequencing to characterize bacterial communities associated with rainfall and, subsequently, used rain-isolated bacterial communities to inoculate lab-grown tomato plants. We then compared the microbiome composition of tomatoes inoculated with (1) bacteria isolated from rain, (2) filter-sterilized rain, and (3) sterile water. Two time points were taken: (1) as soon as plants were dry after inoculation and (2) seven days later. Our analysis revealed a high relative abundance of Proteobacteria on tomato seven days after treatment with rain whereas Actinobacteria and Firmicutes were reduced significantly. At the family level, Pseudomonadaceae, Enterobacteriaceae, Burkholderiaceae and Oxalobacteriaceae present in rain increased the most in relative abundance while Streptomycetaceae, Xanthomonadaceae and Sphingomonadaceae  decreased in abundance. We further performed metagenomic sequencing of rain samples and tomatoes treated with rain on day zero and day seven to start identifying bacteria in rain that efficiently colonize tomato at the strain level. This study provides preliminary evidence that some bacterial taxa present in rain efficiently colonize plant leaves. The question of the relative importance of rain compared to soil as source of the leaf microbiome cannot be answered yet and will be discussed in the context of existing literature. 

 

Andrew Muchlinski, Department of Biological Sciences Formation and function of volatile terpene metabolites in model grasses
Plants sustain intimate relationships with diverse microbiota that contribute to plant productivity and resilience.  Specific plant traits that define the composition of microbial communities by “habitat” filtering remain poorly understood. As microbial community composition can be plant organ specific, a mechanism for selection of particular microbes to niches throughout the plant must exist. Plant derived volatile organic compounds (VOCs), such as terpenoids, are known to affect the colonization of aboveground tissues by epiphytic bacteria. However, the role of VOCs in plant-microbe interactions belowground is limited. We investigate the function of volatile terpenoids as chemo-selective factors in roots using the model grasses Switchgrass and Setaria. We found that both plants accumulate the volatile monoterpenoid borneol in roots; which is likely to affect microbial community composition based on multiple known functions as a microbial growth inhibitor and unique bacterial carbon source. To determine the influence of borneol on the switchgrass root microbiome, we first characterized the terpene synthase (TPS) gene family of switchgrass and associated enzyme function with constitutive and induced VOCs. Within the TPS-a subfamily, we have identified PvTPS04 as terpene synthase that forms borneol as a major product in vitro. Using an RNAi based approach we developed knock-down lines of PvTPS04, and the previously identified borneol synthase gene from Setaria (SvTPS04), to reduce the production of borneol and determine modifications of the root microbiome. While analysis of RNAi lines continues for switchgrass, preliminary evidence from Setaria suggests that a reduction in borneol levels disrupts the “core” microbiome in the endosphere of Setaria roots. Future work will seek to determine taxa specific changes in the microbial community as a result of compound reduction. We expect that our studies underscore the important role of terpenoids as host-specific chemical factors in establishing and maintaining microbial communities in plant roots.

 

Holly Packard, Department of Biological Sciences |  Confirming the essential role of select transcription factors in the phytopathogen Pantoea stewartii during in planta growth through reverse genetics
Pantoea stewartii subsp. stewartii is a bacterial phytopathogen that causes Stewart’s wilt disease in corn.  Genes involved in leaf water-soaking symptoms and xylem biofilm formation are known factors important to the pathogenesis of P. stewartii.  However, much remains to be discovered about in planta specific requirements for the survival and virulence of wilt-disease causing bacteria like P. stewartii.  Previous work in our lab utilized RNA-Seq and Tn-Seq approaches to analyze the wild-type P. stewartii transcriptome expressed in planta and the ability of a library of P. stewartii mutants to survive in the xylem, respectively.  Bioinformatics analysis of these datasets has identified numerous genes of interest that are hypothesized to play an essential role for P. stewartii within the xylem.  Genes encoding two annotated transcription factors, with predicted roles controlling regulons associated with iron-sulfur cluster biosynthesis, were selected for further work to serve as controls for methods development: nsrR (nitric oxide stress response) and iscR (iron-cluster assembly).  Other annotated, but unnamed, transcriptional regulators were also selected to elucidate their role controlling phenotypic outputs important for P. stewartii during infection.  Reverse genetics approaches are underway to generate deletion and complementation strains of each chosen gene. These mutant strains will then be tested via in planta assays to evaluate virulence and colonization capabilities.  Already, NsrR has been confirmed to play a role in P. stewartii virulence.  Ultimately, this work will broaden our understanding of the regulatory networks being employed by the bacteria in planta and may reveal possible disease intervention strategies.

 

Brett Shelley, School of Plant and Environmental Sciences |  Re-Characterization of the Amino Acid Permease I from Arabidopsis: A New Role in Amino 

Plants require nitrogen for many biochemical processes, including protein synthesis and signaling.  While extensive work has described nitrate-induced responses, little is known about amino acid signaling pathways involved in amino acid homeostasis.  Still less is known about how plants sense and respond to external amino acids.  Previous studies showed that exogenous application of glutamine and glutamate alter root architecture, but how these amino acids are sensed remains to be elucidated.  Forward genetic screens for Arabidopsis thaliana mutants resistant to toxic concentrations of amino acids identified the gene that encodes the Amino Acid Permease I (AAP1) protein, involved in the uptake of neutral amino acids across membranes.  The same forward genetic screen identified the phenylalanine insensitive mutant (pig1) mutant, which displays normal amino acid uptake, but is characterized by a deregulation of amino acid metabolism and the accumulation of free amino acids.  Unexpectedly, the Pilot lab recently showed that the pig1-1 mutation is in the AAP1 gene. Other aap1 mutants isolated in the Pilot Lab exhibit amino acid tolerance, while showing either wild type or suppressed amino acid uptake by the roots, suggesting that they might also display, similar to pig1-1, a deregulated metabolism. These dysfunctional mutants thus challenge our current understanding that the role of AAP1 in roots is exclusively amino acid import. Rather, we present evidence that AAP1 could also be involved in regulating amino acid homeostasis via a sensing function.

Kunru Wang, School of Plant and Environmental Sciences  |  Cysteine protease RD21A is required for the drought-induced resistance in Arabidopsis
As one major abiotic stress, drought could significantly reduce crop yields. It has recently been revealed that drought stress also has a strong influence on the plant immunity. However, the molecular mechanism of the drought-regulated immunity has not been well studied. We demonstrated that drought-induced gene RD21A, encodes a cysteine protease, which plays a key role in the drought-induced immunity. The well-watered wild type Arabidopsis (Col-0) plants were more susceptible to a coronatine defective strain DB29 of Pseudomonas syringae pv. tomato DC3000 (Pst-DC3000(DB29)) than that have a temporary drought treatment. However, this drought-induced defense was compromised in the rd21a mutant background. Yeast two-hybrid and co-immunoprecipitation assays uncovered that RD21A interacts with ubiquitin E3 ligase SINAT4. Transient expression results indicated that RD21A could be degraded by SINAT4 in vivo. Consistent with the phenotype of null mutant rd21a, the overexpression of SINAT4 also compromised the drought-induced immunity to Pst-DC3000 (DB29). Furthermore, we also demonstrated that bacterial type III effector AvrRxo1 delivered by Pst-DC3000 (DB29) interacts with both SINAT4 and RD21A in vivo. Protease enzyme assays demonstrated that AvrRxo1 could enhance the E3 enzyme activity of SINAT4, and facilitate the degradation of RD21A in vivo. These results highlight RD21A has a key role in the drought-induced immunity, which can also be targeted by the pathogen virulence effectors.

February 15, 2019 
10:15 am - 12:00 pm
Assembly Hall, Inn at Virginia Tech 

Participant List
Cridland, Caitlin
Mechan, Marco E.
Muchlinski, Andrew
Packard, Holly
Shelley, Brett
Wang, Kunru