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

Document Type : Research Article

Authors

1 University of Zabol

2 Yazd

3 Gorgan

4 Zabol

Abstract

Introduction: 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 S-adenosylmethionine synthase (SAMS) was investigated in both susceptible and tolerant maize genotypes against MDMV at different times after inoculation by qRT-PCR.
 Material and Methods: 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.
Results and Discussion: 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.
Conclusion: 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.

Keywords

References

1. Agrios G. 2005. Plant Pathology. 5th Edition. Academic press, USA.
2. Adhikari T.B., Balaji B., Breeden J., and Goodwin S.B. 2007. Resistance of wheat to Mycosphaerella graminicola involves early and late peaks of gene expression. Physiological and Molecular Plant Pathology, 71: 55-68.
3. Beaulieu A., Williams N., and Patience J. 2009. Response to dietary digestible energy concentration in growing pigs fed cereal grain-based diets. Journal of Animal Science, 87: 965-976.
4. Campo S., Peris-Peris C., Montesinos L., Penas G., Messeguer J., and San Segundo B. S. 2012. Expression of the maize ZmGF14-6 gene in rice confers tolerance to drought stress while enhancing susceptibility to pathogen infection. Journal of Experimental Botany, 63(2): 983-99.
5. Chen F., Li Q., Sun L., and He Z. 2006. The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress. DNA Research, 13: 53–63.
6. Cheng J., Chen J., and Chen J. 2002. The complete sequence of a Sugarcane mosaic virus isolate causing Maize dwarf mosaic disease in China. Journal of Science in China, 45: 322-330.
7. Chevalier D., Morris E.R., and Walker J.C. 2009. 14-3-3 and FHA domains mediate phosphoprotein interactions. Annual Review of Plant Biology, 60: 67-91.
8. Converse R.H., and Martin R.R. 1990. ELISA methods for plant viruses. APS Press. pp. 179-196.
9. Denison F. C., Paul A. L., Zupanska A. K., and Ferl R. J. 2011. 14-3-3 proteins in plant physiology. Seminars in Cell and Developmental Biology, 22:720–727.
10. Dietz K. J. 2008. Redox signal integration: from stimulus to networks and genes. Plant Physiology, 133: 459-468.
11. Fomenko D.E., Koc A., Agisheva N., Jacobsen M., Kaya A., Malinouski M., Rutherford J.C., Siu K.C., Jin D.Y., Winqe D.R., and Gladyshev V.N. 2011. Thiol peroxidases mediate specific genome-wide regulation of gene expression in response to hydrogen peroxide. Proceedings of the National Academy of Sciences, 108: 2729-2734.
12. Garcia-Arenal F., Fraile A., and Malpica J.M. 2001. Variability and genetic structure of plant virus populations. Annual Review of Phytopathology, 39: 157-186.
13. Gomez-Gomez L., and Carrasco P. 1998. Differential Expression of the S-Adenosyl-L-Methionine Synthase Genes during Pea Development. Plant Physiology, 117: 397–405.
14. Gucciardo S., Wisniewski J., Brewin N., and Bornemann S. 2006. A germin-like protein with superoxide dismutase activity in pea nodules with high protein sequence identity to a putative rhicadhesin receptor. Journal of Experimental Botany, 1-11.
15. Godfray H.C.J., Beddington J.R., Crute I.R., Haddad L., Lawrence D., Muir J.F., Pretty J., Robinson S., Thomas S.M., and Toulmin C. 2010. Food security: the challenge of feeding 9 billion people. Science, 327: 812-818.
16. Cueto-Ginzo I., Serrano L., Sin E, Rodriguez R., Morales J., Lade S., Medina V., and Achon M. 2016. Exogenous salicylic acid treatment delays initial infection and counteracts alterations induced by Maize dwarf mosaic virus in the maize proteome. Physiological and Molecular Plant Pathology, 96: 47-59.
17. Hao Z., Wang L., and Tao R. 2010. Defense genes and antioxidant enzymes in stage-dependent resistance to rice neck blast. Journal of Plant Pathology, 92(3): 747-752
18. Hao Z., Wang L., Huang F., and Tao R. 2012. Expression of defense genes and antioxidant defense responses in rice resistance to neck blast at the preliminary heading stage and full heading stage. Plant Physiology and Biochemistry. 57: 222-230.
19. Haslekas C., Viken M.K., Grini P. E., Nygaard V., Nordgard S.H., Meza T.J., and Aalen R.B. 2003. Seed 1-Cysteine Peroxiredoxin Antioxidants Are Not Involved in Dormancy, but Contribute to Inhibition of Germination during Stress. Plant Physiology, 133: 1148–1157.
20. Huang Z., Yeakley J.M., Garcia E.W., Holdridge J.D., Fan J.B., and Whitham S.A. 2005. Salicylic acid-dependent expression of host genes in compatible Arabidopsis -virus interactions. Plant Physiology, 137: 1147–1159.
21. Konagaya K.I., Matsushita Y., Kasahara M., and Nyunoya H. 2004. Members of 14-3-3 protein isoforms interacting with the resistance gene product N and the elicitor of Tobacco mosaic virus. Journal of General Plant Pathology, 70: 221–231.
22. Lu G., DeLisle A. J., Vetten N.C., and Ferl R.J. 1992. Brain proteins in plants: An Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA binding complex. Proceedings of the National Academy of Sciences, 89: 11490–11494.
23. Ludmerszki E., Almasi A., Racz I., Szigeti Z., Solti A., Olah C., and Rudnoy S. 2015. S-methylmethionine contributes to enhanced defense against Maize dwarf mosaic virus infection in maize. Brazilian Journal of Botany, 38(4):771–782.
24. Manosalva P.M., Davidson R.M., Xiaoyuan Zhu B.L., Hulbert S.H., Leung H., and Leach J. N. 2009. A Germin-Like Protein Gene Family Functions as a Complex Quantitative Trait Locus Conferring Broad-Spectrum Disease Resistance in Rice. Plant Physiology, 149: 286- 296.
25. Oh C.S., Pedley K.F., and Martin G.B. 2010. Tomato 14-3-3 protein 7 positively regulates immunity-associated programmed cell death by enhancing protein abundance and signaling ability of MAPKKK α. The Plant Cell, 22:260-272.
26. Park Ch.J., An J., Shin Y., Kim K., Lee L., and Paek K. 2004. Molecular characterization of pepper germin-like protein as the novel PR-16 family of pathogenesis-related proteins isolated during the resistance response to viral and bacterial infection. Planta, 219: 797–806.
27. Patnaik D., and Khurana P. 2001. Germin and Germin like protein: an overview. Indian Journal of Experimental Biology, 39:191-200.
28. Pfaffl M.W. 2001. A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Research, 29:2002–2007.
29. Ravanel S., Gakiere B., Job D., and Douce R. 1998. The specific features of methionine biosynthesis and metabolism in plants. Proceedings of the National Academy of Sciences, 95: 7805–7812
30. Rose R., Erdmann S., Bovens S., Wolf A., Rose M., Hennig S., Waldmann H., and Ottmann C. 2010. and Identification and structure of small molecule stabilizers of 14-3-3 protein interaction. Angewandte Chemie International Edition, 49: 4129-4132.
31. Shukla D.D., Jilka J., Tosic M., Ford R.E., Toler R.W., and Langham M.989. Taxonomy of potyviruses infecting maize, sorghum and sugarcane in Australia and the United States as determined by reactivities of polyclonal antibodies directed towards virus-specific N-termini of coat proteins. Phytopathology, 79: 223-229.
32. Takusagawa F., Kamitori Sh., Misaki Sh., and Markham G.D. 1996. Crystal Structure of S Adenosylmethionine Synthetase. The Journal of Biological Chemistry, 271(1): 136–140.
33. Tobias I., Bakardjieva N., and Palkovics L. 2007. Comparison of Hungarian and Bulgarian isolates of Maize dwarf mosaic virus. Journal of Cereal Research Communications, 35(4): 1643-1651.
34. Tripathi B.N., Bhatt I., and Dietz K.J. 2009. Peroxiredoxins: A less studied component of hydrogen peroxide detoxification in photosynthetic organisms. Protoplasma, 235: 3-15.
35. Trzmiel K., and Jezewska M. 2008. Identification of Maize dwarf mosaic virus in Maize in Poland. Plant Disease, 92(6): 981-992. Abstract.
36. Uzarowska A., Dionisio G., Sarholz B., Piepho H.P., Xu M., Ingvardsen C.R., Wenzel G., and Lubberstedt T. 2009. Validation of candidate genes putatively associated with resistance to SCMV and MDMV in maize (Zea mays L.) by expression profiling. BMC Plant Biology, 9: 1-15.
37. Wei T., Huang T.S., McNeil J.F., Laliberte J.F., Hong J., Nelson R.S., and Wang A. 2010. of the Sequential recruitment endoplasmic reticulum and chloroplasts for plant potyvirus replication. Journal of Virology, 84: 799–809.
38. Williams M.M., and Pataky J.K. 2012. Interactions between maize dwarf mosaic and weed interference on sweet corn. Field Crop Research, 128: 48–54.
39. Wu L., Wang S., Chen X., Wang X., and Wu L. 2013. Proteomic and Phytohormone Analysis of the Response of Maize (Zea mays L.) Seedlings to Sugarcane Mosaic Virus. PLoS ONE, 8(7): 1-17.
40. Zimmermann G., Baumlein H., Mock H., Himmelbach A., and Schweizer P. 2006. The multigene family encoding germin-like proteins of barley: regulation and function in basal host resistance. Plant Physiology, 142: 181–19.
Send comment about this article
CAPTCHA Image

Files

Share

How to cite

Statistics