Investigating the role microRNAs play in the root-knot nematode Meloidogyne incognita’s viability, reproduction, and parasitism in cotton

Abstract

Functions of four microRNAs (miRNAs), min-miR1, min-miR71, min-miR100a, and min-miR-124, were investigated in the parasitic nematode Meloidogyne incognita’s (M. incognita’s) overall behavior and mortality. M. incognita is the most damaging root-knot nematode to the United States’ agriculture industry, negatively impacting crop yield for the cotton plant (Gossypium hirsutum). Much of the current fight against cotton parasites involves chemical pesticides often restricted for environmental concern. Discovering new, biotechnical ways to overcome parasitic infection in cotton plants is more effective in retaining agricultural revenue. Given the conserved nature of these four miRNAs in Caenorhabditis elegans (C. elegans) and M. incognita, I used C. elegans readily available in the lab to test the efficiency of miRNA inhibitors via knockdown of miRNA genes and observed the effects on worms survival/viability. MiRNA inhibitors (anti-miRNAs) were designed according to M. incognita sequence information and used to infect C. elegans at their second juvenile stage that analog the M. incognita parasitic stage J2. Multiple concentrations of each inhibitor were chosen at the levels of 100 nM, 200 nM, 400 nM, and 800 nM with three biological replicates of each. I used a soaking procedure with an optimized transfection solution for the uptake of small miRNAs. Survival data from the treatments was collected, analyzed, and compared. MiRNA gene expression inhibition efficiency was tested using miRNA expression levels via Nanodrop and a routine PCR procedure. Gene expression levels were gathered from these procedures and compared amongst the control and treatments. Survival data thus far has proven that inhibition of the miRNAs affects worms’ survival in a dose-specific manner. Findings from gene expression experiments give way to the success of miRNA gene knockdown in these plant parasites. The results of this experiment can aid in the advancement of biotechnology to combat plant parasitic nematode infection. With these advances, survival of cotton plants should increase in the agricultural realm.