Expression analysis of selected maize primary metabolism genes in response to Cercospora zeina inoculation in the glasshouse

Abstract

Maize is one of the most important grain crops in Africa especially in Southern and sub-Saharan Africa. This crop contributes not only to food security but also to the economy. However, there are factors that affect the development of maize including diseases, which pose as threats to food security. Grey leaf spot (GLS), a foliar disease of maize, is caused by Cercospora zeina, an economically important ascomycete worldwide. It causes over 65% yield losses, thereby being a great threat to food security. This disease was first reported in KwaZulu-Natal province, a GLS hot spot in South Africa in 1988. Strategies such as crop rotation, conventional tillage and fungicides are widely used to manage the progression of GLS. However, these strategies are labour-intensive and expensive for farmers. An alternative cost-effective method is the utilization of host resistance. To better understand the mechanism of host resistance, two primary metabolism genes, isocitrate lyase (ICL) and malate synthase (MS), involved in the glyoxylate pathway, were previously shown to be highly induced by the fungus in a field RNAseq study. This research was aimed at elucidating expression of these two primary metabolism genes. Glasshouse trials were conducted by inoculating the maize genotypes B73 (susceptible) and B73-QTL (resistant) with C. zeina. Genomic DNA was extracted and the fungal CTB7 gene region amplified to confirm that the infection was due to C. zeina. RNA was also extracted, and reverse transcribed to copy DNA (cDNA). The quality of RNA was confirmed by performing a reverse transcriptase PCR (RT-PCR) with banana actin primers as a positive control. Primer pairs flanking an intron in the ICL and MS genes were successfully designed to specifically amplify their respective cDNA. To further confirm these results, the amplified gene products were sequenced using Sanger sequencing platform and thereafter aligned to respective reference genes obtained from NCBI. Overall, ICL and MS genes were successfully amplified from the infected maize genotypes. The ICL amplification needs further optimisation as the bands were not intense to continue with expression analysis. The expression of the two MS transcripts increased at the different progression stages indicated that the glyoxylate pathway plays a role in fungal pathogenicity.