Quantitative and Qualitative Characterization of Plant Endo-bacteriome by Plant Dna-free Sequencing Method

Abstract

Background: Plant endophytic bacteria colonize plants’ internal tissues and interact with plants more closely than do epiphytic or environmental bacteria. However, the community structure of such endophytic bacteria could not be efficiently deciphered, and the microbiota abundance could not be quantified through absolute quantification. Application of 16S rRNA gene sequencing to characterize the plant endophytic community is greatly hindered by the high sequence identities among bacterial 16S rRNA, plant mitochondrial 18S rRNA and chloroplast 16S rRNA genes. This makes it difficult to identify bacterial sequences among total DNAs extracted from plant material.Results: We designed PCR primer sets that are able to specifically amplify bacterial DNAs, even when there are very few bacteria colonizing the plant material. We computationally and experimentally evaluated the specificity, coverage, and accuracy of the newly designed primer sets (322F/796R and 799F/1107R) and two widely used primer sets (338F/806R and 799F/1193R). When applied to a same planting-soil community through next generation sequencing (NGS), the four different primer sets revealed similar high-abundant taxa composition with variation in taxa abundance. Primer sets amplifying the same 16S variable regions generated more comparable sequencing results. When applied to a rice endo-bacteriome, both 799F/1107R and modified 322F-Dr/796Rs (Primer pair 322F/796R with a penultimate-base substitution in 322F) produced plant DNA-free bacterial amplicon libraries. Primer 322F-A/796R was then used through NGS to decipher the rice endo-bacteriomes. The rice root and leaf endo-bacteriomes shared 66.36% OTU identity but enriched different bacterial species. Within the same host genotype and soil type, the root endo-bacteriome was more stable than the leaf endo-bacteriome across individual plants. 322F-A/796R was used through absolute quantitative PCR to quantitate the population size of leaf or root endophytes, which revealed 106–107 and 109–1010 bacteria per gram fresh weight, respectively.Conclusions: This is the first study to develop plant DNA-free bacterial 16S amplification methods. The newly designed primer sets combined with NGS deciphered the rice endo-bacteriome structure, and absolute quantitative PCR quantitated the size of the endobacterial population. The protocols developed here are suitable for various plants, will significantly advance studies on plant endo-bacteriomes.

Phyllosphere Microbiome in Plant Health and Disease

“…The phyllosphere is rich in chlorophyll, and most of the amplicon metagenomics data generated from the phyllosphere are contaminated with the host sequences associated with chloroplast, ribosome, and mitochondria, thus making it difficult to recover the actual genomic information from the sequences. In general, these host-associated reads can be avoided using primer choice or removed during the data processing steps [106]. Recently, scientists have developed a newer method of microbiome analysis using peptide nucleic acids (PNAs) clamping approach and successfully used to suppress the amplification of plant contaminants such as plastid and mitochondrial sequences during PCR amplification [107][108][109].…”

Extension of Plant Phenotypes by the Foliar Microbiome

“…Amplicon metagenomics can be dominated by host chloroplast, mitochondrial, or ribosomal sequences (86). This can be remedied to an extent by primer choice (152), but, in many cases, approaches such as the use of peptide nucleic acids (PNAs) are needed to limit plant amplicons by blocking primer access to unwanted plant target sequences (35). The same problem arises in shotgun metagenomics and transcriptomics, which can be somewhat ameliorated by depletion or enrichment strategies to limit host contamination (113), as well as improvements in bioinformatics tools to identify and remove contaminant sequences (91).…”