Expression Analysis of Some Defense-related Genes in Susceptible and Tolerant Maize Genotypes in Response to Infection of Maize Dwarf Mosaic Virus (MDMV)

Maize (Zea mays L.) is one of the most widely cultivated crops worldwide. More than 40 viruses can infect sweet corn. Maize dwarf mosaic virus (MDMV) is a positive-sense single strand RNA potyviruses that cause significant crop yield loss in susceptible sweet corn varieties. Infected young leaves by MDMV show chlorotic spotting which may eventually turn into a mosaic or mottled pattern. Approximately two-thirds of maize varieties are susceptible to MDMV infection, and even young resistant plants may be infected to the virus at 2-3 leaf stage. These symptoms vary greatly depending on the host genotype, time of infection, and on the strain causing the injection. However, the precise molecular details of maize responses to MDMV infection are largely unknown. In sensitive and tolerant plants during viral infection, the expression level of defense -related genes are altered based on plants ability to recognize pathogen attack. The elevated expression of defense-related genes such as SAMS and G-box factor 14-6 (GF146) leads to production of resistance proteins which are considered as molecular response of maize to variety of biotic stresses. The production of resistance proteins and the enhanced expressions of Peroredoxin, SAMS, G-box factor 14-6 (GF14- 6), and other genes are considered as typical responses in maize varieties. Proteomic analysis in susceptible and tolerant maize seedlings infected by sugarcane mosaic virus showed a high expression of 96 different proteins. In this study, the expression level of some defense genes including Germin like protein (GLP), Peroxiredoxin (Prx), GF14-6 and Sadenosylmethionine synthase (SAMS) was investigated in both susceptible and tolerant maize genotypes against MDMV at different times after inoculation by qRT-PCR. The seed of SC705 cultivar and the number of hybrid number 8 (KLM75010/4-4-1-2-1- 1-1×MO17) were used as susceptible and tolerant cultivar to MDMV, respectively. All the seeds were sterilized with chloramine T (3%), washed three times with distilled water and then grown in a greenhouse under controlled conditions at 25°C. Leaves of infected plants by MDMV showing macroscopic symptoms were homogenized in phosphate buffer (pH 7.2, 0.06 M) and were used for inoculation using carborundum. The leaves of mock-plants were mechanically injured and infected with phosphate buffer. Sampling was done at 5-time intervals including 0, 1, 9, 24 and 72 hours after plant viral infection. Total RNA was extracted from the leaf tissue and further treated with RNase free DNaseI to eliminate any DNA contamination. Reverse transcription reaction was performed using M-MuLV reverse transcriptase. Gene expression analysis was done using qRT-PCR method by an iCycler instrument (iQ5, BioRad, USA) using SYBR Green PCR MasterMix and the relative gene expression was calculated according to ΔΔCT method according to the Pfaffl method. The reaction of PCR was carried out at 50 °C for 2 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 59 ºC for 20 s, and 72 ºC for 20 s. Reactions were finished with a dissociation step, starting at 55°C to 95 °C by increasing 0.5°C per cycle for 80 cycles. Symptoms of infected susceptible and tolerant plants were monitored for 20 days post inoculation (dpi). The success of infection was verified using the ELISA technique. All the susceptible SC705 cultivars showed visible mosaic symptoms at 16 dpi to MDMV while 8-hybrid plants showed mild mosaic symptoms at 19 dpi. Gene expression analysis showed that the expression level of all tested genes was significantly increased in tolerant maize in comparing with susceptible maize plants. One hour after maize inoculation an increase of expression level was seen for all tested genes. Peroxiredoxin and GLP were up-regulated at 1 hpi and then decreased over time. SAM and GF14-6 were up-regulated in the all-time intervals but the highest expression level was noted at 24 hpi. The lowest expression level for all tested genes was observed at 72 hpi. In the other word, the expression levels of SAM and GF14- 6 genes were elevated in a moderate rate with a continuous increase in the infected maize from first time point after inoculation until last time point of sampling. The rapid induction of defense-related genes is required to activate defense mechanisms and respond to against pathogens attacks. In this research, we tested the expression profile of four genes which have been defined as important proteins related to disease and stress signals. Based on these results, we can conclude that tested genes in this research could be suitable as biomarkers for the selection of tolerant or relatively tolerant maize cultivars against MDMV. Among them, GLP gene was more efficient as a screen able marker.