A plant transformation system designed for high throughput genomics inGossypium hirsutumto study root–organism interactions

Abstract

The study of biological processes has been aided greatly by the development of procedures to identify the large numbers of associated genes. However, the ability to study the identified genes experimentally is often impeded by the absence of technologies to perform such functional analyses. Here, a nonaxenic plant transformation system has been developed in Gossypium hirsutum (cotton) for the study of genes associated with root functions and root-organism interactions. The plant transformation system is compatible with modern high throughput plant transformation goals and the processing of large numbers of genes intended for the study of root function in G. hirsutum.

The heterologous expression of aGlycine maxhomolog of NONEXPRESSOR OF PR1 (NPR1) and α-hydroxynitrile glucosidase suppresses parasitism by the root pathogenMeloidogyne incognitainGossypium hirsutum

“…Therefore, each individual transgenic root system functions as an independent transformant line (Tepfer 1984;Matsye et al 2012). The DNA backbone of the engineered pRAP15 contains nucleotide sequences containing both the eGFP and the gene of interest (GOI), each with their own promoter and terminator sequences (Pant, McNeece et al 2015). Thus, roots expressing eGFP will also contain the GOI.…”

“…Overexpression studies are performed using the pRAP15 vector transformation system (Matsye et al 2012;Pant et al 2014;Pant, McNeece et al 2015). The transgenic roots are produced by using Agrobacterium rhizogenes strain 15834 (Pant, McNeece et al 2015). Due to the way A. rhizogenes transfers the DNA cassettes between the left and right borders of the plasmid to the root cell DNA, the subsequent growth and development of the genetically engineered cell into a transgenic root results in the production of a plant that is a genetic mosaic with the shoot being nontransgenic and the root transgenic.…”

“…Overexpression studies are performed using the pRAP15 vector transformation system (Matsye et al 2012;Pant et al 2014;Pant, McNeece et al 2015). The transgenic roots are produced by using Agrobacterium rhizogenes strain 15834 (Pant, McNeece et al 2015).…”

“…The pRAP15 control and engineered vector are used to transform chemically competent A. rhizogenes (Hofgen & Willmitzer 1988;Haas et al 1995) on tetracycline (5 μg/ml) according to Matsye et al (2012). Genetic transformation experiments resulting in heterologous gene expression in G. hirsutum are performed according to Pant, McNeece et al (2015). RNA is extracted from G. hirsutum roots using the UltraClean® Plant RNA Isolation Kit (Mo Bio Laboratories®, Inc.; Carlsbad, CA) and treated with DNase I according to the manufacturer's instructions (Invitrogen®).…”

“…In the analysis presented here, roots that have been shown to express a G. max NPR1 homolog (Gm-NPR1-2) in Gossypium hirsutum (Pant, McNeece 2015) have been infected by M. incognita. The expression of Gm-NPR1-2 in G. hirsutum results in the suppression of the formation of galls, the production of egg sacs, the production of eggs and the development of J2 nematodes as compared to controls lacking the transgene.…”