Progress 01/01/22 to 08/31/23
Outputs
Target Audience:Microbial ecologists, plant biologists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Trained two postdoctoral researchers and one undergraduate researcher How have the results been disseminated to communities of interest?In addition to scientific talks reported in the last progress report, disseminated results at an invited seminar at the University of Pittsburgh What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Results for aims 1-2:The pH was measured on the leaf surface for species with pH variation on its phylloplane from alkaline (Gossypium, pH ~8.8), around neutral (Beta, pH ~7.8), to acidic (Nepenthes, pH ~4.8) on a dry control and in response to each pH treatment (pH 6.5, 4, and 2) to examine pH regulation ability. A comparative transcriptomic analysis was performed with the same multifactorial experiment design. Orthologues annotation across species was done with OrthoFinder. We found specific buffering ability and differentially expressed genes linked to each genus. Gossypium were the only species that showed a strong buffering ability. At the pH 6.5 and pH 4 treatments, they alkalinized phylloplane pH slightly higher than the dry control pH, and even increased, allowing the pH 2 treatment to be around pH 6 in 5 minutes. At the transcriptional level, each species showed specific differentially expressed isoforms, which suggest each of this genus sense and respond to external pH changes in a unique way. Some DE genes related to the response to external pH changes involve ATPase-H+ pumps and ABC transporters. Gene ontology term enrichment has shown transmembrane transport as one of the main categories highly present. Some of the DE genes were involved in other abiotic stresses such as chemical and drought stress. Overall, these results provide insights into the diverse physiological responses different species have to external pH changes and possible mechanisms by which plants can sense and potentially counteract these changes. Results for aim 3:Young leaves were inoculated with a common microbial community sourced from a soil slurry. Leaves were also treated with one of two external pH treatments, pH 6.5 or pH 2. After exposure to the pH treatment, we sonicated the leaves to collect phylloplane microbes and performed total RNA extractions and short-read sequencing. We performed metatranscriptome assemblies with SAMSA2. We found that host species significantly shaped community composition of microbial genes, which all diverged from the source inoculum; however, this was not significantly impacted by pH treatment. Differential expression analysis, however, revealed differentially expressed genes between pH 2 and pH 6.5. Indicator genes analysis also revealed specific functional gene groups significantly associated with each plant species, underlining the importance of the active recruitment done by plant hosts in shaping their phylloplane microbiome. Further analyses are ongoing after obtaining microbial data pulled from the transcriptome data from the aims above in order to investigate the influence of host regulation and external pH changes on the plant endophytic microbial communities, i.e. microbial cells found inside the sequenced leaf tissues. This is leading to further interesting results providing insight into how host physiology shapes their entire microbial communities. Similar to our innovulated phylloplane experiment described for aim 3, we found a significant (even stronger) effect of host species on the community composition of microbial transcripts. In contrast to the results for external microbes, the endophytic communities were also significantly structured by the pH treatment (pH 6.5, 4, or 2) as well as the interaction between host species and pH treatment. Further, we found interesting similarities in the functional genes that were overrepresented on a particular host between the two experiments. For instance, flagellin genes and virulence-associated gene ontogeny terms were overrepresented onBeta vulgarisleaves for both the phylloplane and endophyte data. This suggests that beet leaves, which create much less extreme pH conditions on their leaves relative to the other species in this study, create an environment that are particularly favorable for pathogenic species. This yields implications for the power of extreme phylloplane pH modification (either alkalinization or acidification) in creating a non-conducive environment for pathogens in a shared environment. Results for aim 4:With an undergraduate researcher I mentored, we increased phylogenetic coverage beyond the taxa previously examined during my time at Penn State, and also discovered that environmental heterogeneity can affect phylloplane pH, causing it to deviate away from its typical mean within a species. We took a more indepth look at phylloplane pH regulation in maize (Zea mays), examining whether soil nitrogen addition affects external pH buffering ability. One key finding was that microscale variation in soil pH was significantly correlated to dry phylloplane pH levels, but not wet phylloplane pH (pH after being sprayed with neutral water), suggesting that dry phylloplane pH could be a consequence of passively translocating ions from the soil onto the surface. On the other hand, wet phylloplane pH appears to be more physiologically active, and the pH levels achieved were dependent more on the developmental stage of the plant than any external factors. Several manuscripts are still in progress for this project: one paper on host plant transcriptomic responses entitled "Species-specific phyllosphere responses to external pH change" targeted for submission to Plant Physiology & Biochemistry, and one paper for the associated microbial communities on the phylloplane as well as inside the leaf entitled "The role of host-specific phylloplane pH regulation in shaping gene expression of bacterial communities" targeted for submission to Phytobiomes, as well as a manuscript in the earlier stages of preparation on maize phylloplane pH regulation.
Publications
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Progress 01/01/23 to 08/31/23
Outputs
Target Audience:Microbial ecologists, plant biologists Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Trained two postdoctoral researchers and one undergraduate researcher How have the results been disseminated to communities of interest?Presented results at several invited seminars at the University of Minnesota, Kalamazoo College, Emory University, Western Michigan University, and the University of Kansas. Also presented results at the Ecological Society of America meeting in August 2023. What do you plan to do during the next reporting period to accomplish the goals?Submit manuscripts on plant transcriptomic and microbial metatranscriptomic projects (aims 2-3). Also prepare and submit manuscript on the associated maize phylloplane pH study conducted with REU student (aim 4, in part). Analyze 16S metabarcoding data from trial study of young leaf phylloplane microbiome from multiple Gossypium species grown in common garden. The work on multiple Gossypium species will deepen our knowledge of phylloplane microbiome assembly obtained from the five species in the current manuscript in prep. Because we have discovered that some Gossypium clades alkalinize (A genome and AD allopolyploids) and others do not (D genome, C genome, outgroups), this will be a vital contribution to the original goals of the grant as these data will better allow us to disambiguate the role of phylogeny vs. physiology in microbiome assembly. As we had fairly low success in getting 16S sequences to amplify from our 48 extracted samples, I am requesting an additional 1-year NCE in order to have enough time to repeat this work on Gossypium, collecting a greater number of samples in order to get better between-species comparisons in microbiome composition with sufficient replication. I plan to try two approaches for two different leaf developmental stages to both account for a potentially low PCR amplification success rate (~50% for 16S metabarcoding of young leaves), as well as the added benefit of a cross-stage comparison if both stages yield data. On the one hand, I plan to sample young leaves with a similar method as before, sonicating the samples directly into DNA/RNA Shield in 2mL centrifuge tubes. In this case, I plan to get seeds of the same 14 species from my collaborator Jonathan Wendel, and sample 6 replicates per individual species by growing them to the two-leaf stage in my greenhouse, which will ensure enough young leaves across species, and reduce the prior potential risk of microbe communities changing during the travel time previously needed to get the leaves to my lab. This would yield 84 samples for DNA extraction and attempted 16S metabarcoding. Additionally, I want to continue to utilize the mature plants available in the Wendel greenhouse, and get 12 reps of fully expanded mature leaves (on lower nodes) for these same 14 species. We will attempt a modified protocol for obtaining the microbial communities from the phylloplane as mature leaves are too large for centrifuge tubes, and the cost of the volume of DNA/RNA Shield needed would be prohibitive. Instead, these will be sonicated in Falcon tubes with molecular grade sterile water to recover the microbes. This would yield 168 samples for DNA extraction. The focus will be on 16S metabarcoding as for the young leaves, but we might get ITS primers as well in order to test a small subset to confirm if young leaves are indeed relatively free of fungi as compared to mature leaves. Mature leaves likely have higher cell densities and overall microbial diversity, but may involve more stochasticity as well in the outcome of their community assembly. In combination with the data from young leaves, we will get a clearer sense of the extent of the influence of the hostplant.
Impacts
What was accomplished under these goals?
In the past 12 months, I have hired two postdoctoral researchers to join my nascent lab group, and with their help, we have made substantial strides in the analysis of the data I've collected for aims 2 & 3 (plant transcriptomics and microbial metatranscriptomics, respectively). This has led to a deeper understanding of the influence of external pH changes across the five study species, and how these hostplants in turn influence the gene expression of microbial communities on their leaf surfaces via differences in phylloplane pH regulation. The results of these aims are currently being written up in two separate manuscripts which are nearing completion; I will append the two draft abstracts below, which will explain more specific details of our results. We plan to submit the manuscript tentatively entitled "Species-specific phyllosphere responses to external pH change" to the journal Plant Physiology and Biochemistry, and the one entitled "The role of host-specific phylloplane pH regulation in shaping gene expression of bacterial communities" to Phytobiomes. In addition to these two aims, further progress was made in aim 4 (exploration of natural variation in phylloplane pH across many taxa) in work with an undergraduate student I mentored as part of the Kellogg Biological Station REU program. We increased phylogenetic coverage beyond the taxa previously examined during my time at Penn State, and also discovered that environmental heterogeneity can affect phylloplane pH, causing it to deviate away from its typical mean within a species. We took a more in-depth look at phylloplane pH regulation in maize (Zea mays), examining whether soil nitrogen addition affects external pH buffering ability. One key finding was that microscale variation in soil pH was significantly correlated to dry phylloplane pH levels, but not wet phylloplane pH (pH after being sprayed with neutral water), suggesting that dry phylloplane pH could be a consequence of passively translocating ions from the soil onto the surface. On the other hand, wet phylloplane pH appears to be more physiologically active, and the pH levels achieved were dependent more on the developmental stage of the plant than any external factors. We are also beginning to prepare a manuscript for publication from this work. In yet another project related to the aims of this grant, my postdocs and I are delving deeper into understanding interspecific pH variation within the genus Gossypium and how/whether that influences their phylloplane microbiomes. With our collaborator Jonathan Wendel at Iowa State University, we sampled 14 Gossypium species (and the outgroup Kokia drynarioides), with ~3 young leaves from each, for a total of ~48 leaf samples sonicated to get microbes into DNA/RNA Shield, which we then performed DNA extractions on for 16S and ITS metabarcoding. Unfortunately, the ITS primers could not be amplified, likely due to a lack of substantial fungal DNA on the young leaves. Further only half of the extractions could successfully be amplified for 16S. We have recently sent out these 24 successful samples for sequencing at the Michigan State University Genomics Core. Abstracts for main manuscripts from aims 2 and 3, respectively: Species-specific phyllosphere responses to external pH change The leaf surface (the phylloplane) is the first point of contact in the interaction between plant and environment aboveground. These plant-environment interactions can involve pH changes, such as when it is in contact with pesticide application, acid rain, microbes, and pests. It has been shown that a plant can modify its pH on the phylloplane, and this buffering ability is plant species-specific. Genes and/or pathways involved in the responses to external pH changes, and how they can be related to other abiotic and biotic stress signaling pathways haven't been described. The pH was measured on the leaf surface for species with pH variation on its phylloplane from alkaline (Gossypium, pH ~8.8), around neutral (Beta, pH ~7.8), to acidic (Nepenthes, pH ~4.8) on a dry control and in response to each pH treatment (pH 6.5, 4, and 2) to examine pH regulation ability. A comparative transcriptomic analysis was performed with the same multifactorial experiment design. Orthologues annotation across species was done with OrthoFinder. We found specific buffering ability and differentially expressed genes linked to each genus. Gossypium were the only species that showed a strong buffering ability. At the pH 6.5 and pH 4 treatments, they alkalinized phylloplane pH slightly higher than the dry control pH, and even increased, allowing the pH 2 treatment to be around pH 6 in 5 minutes. At the transcriptional level, each species showed specific differentially expressed isoforms, which suggest each of this genus sense and respond to external pH changes in a unique way. Some DE genes related to the response to external pH changes involve ATPase-H+ pumps and ABC transporters. Gene ontology term enrichment has shown transmembrane transport as one of the main categories highly present. Some of the DE genes were involved in other abiotic stresses such as chemical and drought stress. The role of host-specific phylloplane pH regulation in shaping gene expression of bacterial communities Plants can differ in their ability to regulate pH conditions on their leaf surfaces (phylloplane), ranging from hyper-acidic carnivorous plants to hyper-alkaline Malvaceae. These physiological differences also extend to their ability to buffer against external pH changes (e.g., acid rain). Given the importance of soil pH to the rhizosphere microbiome, phylloplane pH regulation may be important in shaping leaf microbiome, however, the potential consequences of this were previously unknown. In this study, we took a metatranscriptomic approach to examine phylloplane bacteria on five plant species that differ in phylloplane regulation (including species of Nepenthes and Gossypium at the acidic and alkaline extremes, respectively). Young leaves were inoculated with a common microbial community sourced from a soil slurry. Leaves were also treated with one of two external pH treatments, pH 6.5 or pH 2. After exposure to the pH treatment, we sonicated the leaves to collect phylloplane microbes and performed total RNA extractions and short-read sequencing. We performed metatranscriptome assemblies with SAMSA2. We found that host species significantly shaped community composition of microbial genes, which all diverged from the source inoculum; however, this was not significantly impacted by pH treatment. Differential expression analysis, however, revealed differentially expressed genes between pH 2 and pH 6.5. Indicator genes analysis also revealed specific functional gene groups significantly associated with each plant species, underlining the importance of the active recruitment done by plant hosts in shaping their phylloplane microbiome.
Publications
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