Phenotypic and molecular evaluation of cotton hairy roots as a model system for studying nematode resistance

Authors: Wubben, Martin; Callahan, Franklin; Triplett, Barbara; Jenkins, Johnie

Submitted to: Plant Cell Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/22/2009
Publication Date: 8/1/2009
Citation: Wubben, M., Callahan, F.E., Triplett, B.A., Jenkins, J.N. 2009. Phenotypic and Molecular Evaluation of Cotton Hairy Roots as a Model System for Studying Nematode Resistance. Plant Cell Reports. 28:1399-1409.

Interpretive Summary: The root-knot and reniform nematodes are serious pests of Upland cotton in the United States and abroad. There has been little research regarding the cellular mechanisms that control the resistance that has been identified in various cotton genotypes. This lack of research is partly due to the root parasitic nature of the nematode and to the difficulty in producing transgenic cotton for the purpose of determining gene function. Therefore, we investigated whether cotton hairy roots, generated by Agrobacterium rhizogenes infection of cotyledons, could serve as a model system for studying the cotton-nematode interaction. Hairy roots can be grown indefinitely in tissue culture and are more amenable to genetic manipulation compared to whole plants. We determined that both root-knot and reniform nematode will infect and reproduce on cotton hairy roots derived from a susceptible cotton line (DP90). Furthermore, we found that hairy roots generated from resistant cotton lines (M315 and GB713) maintained their resistance in hairy roots. In addition to nematode inoculation experiments, a series of molecular assays were performed including gene expression and protein analyses. Our results show that cotton hairy roots can be used to maintain root-knot and reniform nematode inoculum under sterile conditions; however, caution is warranted when interpreting data collected using hairy roots because of significant variation in gene expression and protein levels between hairy root lines.

Technical Abstract: The cellular mechanisms that mediate resistance of allotetraploid cotton (Gossypium spp.) to root-knot nematode (Meloidogyne incognita) and reniform nematode (Rotylenchulus reniformis) are poorly understood. Here, Agrobacterium rhizogenes-induced hairy roots were investigated as a possible research platform for studying RKN and reniform nematode parasitism of cotton in tissue culture. Morphology, growth rate, nematode infection, and gene expression of hairy root cultures initiated from M315 (RKN-resistant), GB713 (reniform-resistant), and DP90 (susceptible) genotypes were compared. Variation in hairy root morphology and growth rate was observed across all cultures; however, this variation was not correlated with basal transcript levels of the A. rhizogenes rolB and aux2 genes. RKN and reniform nematode successfully infected and reproduced on DP90 hairy roots in tissue culture. In contrast, no RKN reproduction was observed on M315 hairy roots. Likewise, the GB713 culture supported significantly fewer reniform nematode females compared to DP90. Basal transcript and protein levels of MIC3, a RKN resistance-associated gene, were compared between native and hairy roots to assess possible transformation-related effects on root gene expression. Although uninfected native roots of RKN-resistant M315 showed low basal MIC3 transcript levels and undetectable protein, M315 hairy root lines expressed elevated and variable levels of MIC3 transcript and protein. DP90 hairy roots did not show elevated levels of either MIC3 transcript or protein. Our results indicate that cotton hairy roots can be used to propagate RKN and reniform nematode under sterile conditions; however, comparisons between nematode-resistant and -susceptible hairy root cultures may be confounded by A. rhizogenes transformation-related changes in root gene expression.