Accelerating Complete Phytoplasma Genome Assembly by Immunoprecipitation-Based Enrichment and MinION-Based DNA Sequencing for Comparative Analyses.

Choon Meng Tan,Chih-Horng Kuo,Jun-Yi Yang,Yuan-Yu Chien,Chien-Jui Wang,Cheng-Wei Wang,Yu-Chen Lin,Lin Chou,Yi-Ching Chiu,Jian-Rong Li

doi:10.3389/fmicb.2021.766221

Abstract

Phytoplasmas are uncultivated plant-pathogenic bacteria with agricultural importance. Those belonging to the 16SrII group, represented by ‘Candidatus P. aurantifolia’, have a wide range of plant hosts and cause significant yield losses in valuable crops, such as pear, sweet potato, peanut, and soybean. In this study, a method that combines immunoprecipitation-based enrichment and MinION long-read DNA sequencing was developed to solve the challenge of phytoplasma genome studies. This approach produced long reads with high mapping rates and high genomic coverage that can be combined with Illumina reads to produce complete genome assemblies with high accuracy. We applied this method to strain NCHU2014 and determined its complete genome sequence, which consists of one circular chromosome with 635,584 bp and one plasmid with 4,224 bp. Although ‘Ca. P. aurantifolia’ NCHU2014 has a small chromosome with only 471 protein-coding genes, it contains 33 transporter genes and 27 putative effector genes, which may contribute to obtaining nutrients from hosts and manipulating host developments for their survival and multiplication. Two effectors, the homologs of SAP11 and SAP54/PHYL1 identified in ‘Ca. P. aurantifolia’ NCHU2014, have the biochemical activities in destabilizing host transcription factors, which can explain the disease symptoms observed in infected plants. Taken together, this study provides the first complete genome available for the 16SrII phytoplasmas and contributes to the understanding of phytoplasma pathogenicity.

Highlights

Phytoplasmas are wall-less bacterial pathogens that are known to infect numerous plant species and lead to significant agricultural losses (Gurr et al, 2016; Kumari et al, 2019; Pierro et al, 2019)
To enrich ‘Ca. P. aurantifolia’ NCHU2014 for whole genome sequencing, immunoprecipitation-based purification of phytoplasma cells was conducted using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of ‘Ca. P. aurantifolia’ NCHU2014
Genomic DNA extracted from the immunoprecipitated fraction containing ‘Ca. P. aurantifolia’ NCHU2014 was obtained for PCR examination

Summary

Introduction

Phytoplasmas are wall-less bacterial pathogens that are known to infect numerous plant species and lead to significant agricultural losses (Gurr et al, 2016; Kumari et al, 2019; Pierro et al, 2019). They are parasitic bacteria multiplying exclusively in phloem sieve elements and are transmitted between plants by phloemfeeding insects (Lee et al, 2000; Hogenhout et al, 2008). As an obligate intracellular parasite, phytoplasmas remain some of the most challenging plant pathogens to characterize due to the lack of an axenic culture

Methods

Results

Conclusion