Molecular analyses of the interaction of microbes and marsh grasses  Spartina alterniflora and Phragmites australis Lathadevi K. Chintapenta1; Venkateswara Sripathi2,3; Mollee Crampton2; Gulnihal Ozbay1; Venu (Kal) Kalavacharla2* 1Aquatic and Environmental Sciences Laboratory, College of Agriculture and Related Sciences, Delaware State University, Dover, DE 19901 2Molecular Genetics and Epigenomics Laboratory, College of Agriculture and Related Sciences, Delaware State University, Dover, DE 19901 3College of Agricultural, Life and Natural Sciences, Alabama A&M University, Huntsville, AL 35811 *Center for Integrated Biological and Environmental Research (CIBER), Delaware State University, Dover, Delaware, 19901, USA Abstract: Plant microbiomes play a key role in plant survival, as microbial interactions contribute to plant growth and vigor by helping to tolerate stress. These interactions are triggered by signaling molecules which regulate gene expression. Plants from marsh environments are constantly exposed to stress due to variations in hydrology and salinity levels. The main focus of this research is to explore the microbial community associated with native and invasive marsh grasses and the differences in gene expressions of these grasses. In the long term, we aim to understand how microorganisms assist marsh grasses in tolerating abiotic stress conditions. The underlying objective will be achieved by microbial composition profiling and Illumina sequencing of plant transcriptomes. Symbiotic microbial diversity was high in Spartina soils and roots particularly alpha, beta, and delta proteobacteria. Several genes known to be upregulated during salt stress were identified from the transcriptomes of Spartina and Phragmites leaves. Results from this study will help distinguish salt stress genes which are being influenced by microbes. The future prospects of this study will provide better insight on the role of microbes in terrestrial plant survival under extreme environmental conditions. Introduction Microorganisms associated with plants help in regulating the plant immune system which affects plant growth and development, especially in stress conditions. Bacteria and fungi communicate with their plant hosts by producing chemicals, and plants in turn respond to these signals by adjusting their growth and defense mechanisms (Randy et al, 2009). In the long run, this research aims to address the role of microbes in the salt tolerance mechanisms of Spartina alterniflora and Phragmites australis. Results Alpha, beta and gamma proteobacteria along with cyanobacteria and Frankia species in actinobacteria form symbiotic associations in plants. Actinobacteria and cyanobacteria were dominant in the tissues of Spartina. The sequences of Phragmites matched with Camelina sativa, Zea mays, Oryza sativa. Transcripts identified- 42,500. All the proteobacterial groups were dominant in the stem and soil samples of Spartina. The beta proteobacteria were abundant only in the roots and leaves of Phragmites (Fig.3). Fig. 7: Gene Ontology of Pragmites australis leaves Genes induced during salt stress The genes involved in ion transport, osmolyte biosynthesis and several house keeping genes are known to be upregulated during salt stress in S. alterniflora (Baisakh et al, 2008). Objectives 1. Identification of dominant microbial groups associated with two marsh grass species (S. alterniflora and P. australis). 2. Transcriptome analysis of root and leaf tissues of marsh grasses collected from the native environment. Materials and Methods Leaves, stems, roots and soils surrounding the plant roots were collected from S. alterniflora and P. ausralis of Blackbird Creek in Delaware. Fig. 8: Salt stress induced genes in Spartina alterniflora leaves from public databases In these marsh grasses more than 50% of the salt stress induced genes belonged to signal transduction and transcriptional machinery. Ribosomal proteins known to be up- and down regulated during salt stress (Sahi et al, 2006) were higher in Spartina (12%) than Phragmites (5%). Zinc finger (C2H2) family protein, a transcriptional repressor under high salinity (Sakamoto et al, 2004) was expressed in both species whereas, Histone deacetylase HDA2 was absent. Genes involved in Reactive Oxygen Species (ROS) pathways are influenced by microbes Spooner and Yilamez, 2011). ROS genes were high in Phragmites (15%) than Spartina (6%) while the symbiotic microbial population is more in Spartina. Current and future studies involve comparative analysis of the plant transcripts with the Microbial Genome Database in order to understand if symbiotic microbe associated genes are present. Fig. 3: Symbiotic bacteria taxa in the plant samples Fig. 1: Spartina alterniflora Phragmites australis Microbial community Analysis DNA isolation: Zymo Research DNA kit. Two biological and six technical replicates were Used. Microbial composition Profiling: targeted 16s rRNA gene. Next generation sequencing: Illumina Miseq platform (Fig. 2a) at Zymo Research, Irvine, CA Plant Transcriptome Analysis RNA isolation: Trizol method. cDNA libraries were constructed and sequenced on the Illumina HiSeq 2500 platform (Fig. 2b), at the Delaware Biotechnology Institute. Fig. 5: VAM fungi observed in stained Spartina roots, under 10X and 40x magnification Fig. 4: Alphararefaction curves for bacterial communities S. alterniflora (native marsh grass) had abundant symbiotic microbes than P. australis (invasive marshgrass). Alpha diversity analysis was performed on the Operational Taxonomic Units (OTUs) or species to determine the species richness. Rarefaction plots show that bacterial diversity was high in soil samples than plant tissues (Fig. 4). Spartina sequences matched with Oryza sativa, Zea mays, Setaria italica. Transcript identified- 13,250. Fig. 2a: Microbial community profiling References Baisakah et al, (2008). Primary responses to salt stress in a halophyte, smooth cordgrass (Spartina alterniflora Loisel.) Funct Integr Genomics. Ralee Spooner and Özlem Yilmaz (2011). The Role of Reactive-Oxygen-Species in Microbial Persistence and InflammationInt. J. Mol. Sci. 2011, 12, 334-352; Randy et al, (2009). The role of microbial signals in plant growth and development, Plant Signaling & Behavior 4:8, 701-712. Sahi et al, (2006) Salt stress response in rice: genetics, molecular biology, and comparative genomics. Funct Integr Genomics 6:263–284. Sakamoto et al, (2004) Arabidopsis cys2/his2-type zinc-finger proteins function as transcription repressors under drought, cold stress. Plant Physiol 136:2734–2746. a Acknowledgments We thank NSF-EPSCoR program (EPS-1301765) USDA for their funding support. DNREC for their assistance with sampling. We also thank our undergraduate and graduate students and other lab members at Delaware State University for their constant support. Fig. 6: Gene Ontology of Spartina alterniflora leaves Fig. 2b: RNA sequence analysis