Cochliobolus lunatus abundantly produces four-celled conidia at high temperatures (>30 °C) and under suitable conditions; the fungus colonizes potato (Solanum tuberosum L.) cultivars by adopting different invasion strategies at the microscopic level. Long-lasting defence during infection requires an upsurge in proteome changes particularly pathogenesis-related proteins chiefly under the control of nonexpresser of pathogenesis-related proteins. In order to gain molecular insights, we profiled the changes in proteome and potato nonexpresser of pathogenesis-related proteins (StNPR1) during the infection process. It is found that C. lunatus significantly (P < 0.05) suppressed the host functional proteome by 96 h after infection (hai), principally, affecting the expression of ribulose bisphosphate carboxylase enzyme, plastidic aldolase enzyme, alcohol dehydrogenase 2 and photosystem II protein prior to the formation of brown-to-black leaf spot disease. Strongest host response was observed at 24 hai hallmarked by 307 differentially expressed peptide spots concurring with the active phase of production of penetrating hyphae. Additionally, C. lunatus differentially down-regulated StNPR1 transcript by 8.19 fold by 24 hai. This study is the first to elucidate that C. lunatus transiently down-regulates the expression of StNPR1 at the onset of infection, and as a whole, infection negatively affects the expression of proteome components involved in photosynthesis, carbon fixation and light assimilation. This study contributes towards better understanding of the mechanism underlining the invasion strategies of C. lunatus.
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This research was jointly supported by The World Academy of Sciences (TWAS), Trieste, Italy and the Department of Biotechnology, Government of India (DBT/TWAS PG fellowship No. 3240223450) and Alexander von Humbolt (AvH) foundation. The authors thank DK Hore for proofreading the manuscript.
Communicated by Erko Stackebrandt.
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A 2-D proteome map of potato leaves at 24 hai of C. lunatus. Spots of interest with fold expression >3 are indicated with arrows. The immobilized pH gradient scale and Precision Plus Protein™ WesternC™ Standards are shown (TIFF 227 kb)
A 2-D proteome map of potato leaves at 72 hai of C. lunatus. Spots of interest with fold expression >3 are indicated with arrows. The immobilized pH gradient scale and Precision Plus Protein™ WesternC™ Standards are shown (TIFF 219 kb)
Reverse transcriptase PCR for 26S rRNA endogenous control following cDNA synthesis from total RNA. The reaction mixture consisted of 0.5 μl of 10 mM dNTPs, 19.8 μl DEPC H2O, 2.5 μl of 10X buffer, 0.5 μl of 20 pmol/μl forward, 0.5 μl of 20 pmol/μl reverse primers, 0.2 μl of 5U/ul TaqPol, and 1 μl of cDNA. The PCR conditions were: 94 °C for 3 min initial denaturation, 35 cycles of 94 °C for 30 s, 54 °C for 40 s, 72 °C for 1 min and a final extension at 72 °C for 7 min. Agarose gel electrophoresis showing a 500 bp of 26S rDNA amplicon for samples extracted at different time points. A24, A48, A72 and A96 are cDNA for plants treated with sterile water. Ctlr is control plant not treated with any substance. EA24, EA48, EA72 and EA96 are cDNA for plants challenged with C. lunatus (TIFF 78 kb)
Primers set for genes that encode peptide spots, used for validating the expression of significantly alter peptide spots (XLSX 10 kb)
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Louis, B., Waikhom, S.D., Jose, R.C. et al. Cochliobolus lunatus down-regulates proteome at late stage of colonization and transiently alters StNPR1 expression in Solanum tuberosum L.. Arch Microbiol 199, 237–246 (2017). https://doi.org/10.1007/s00203-016-1297-2
- Two-dimensional electrophoresis
- Western blotting
- MALDI-TOF/TOF MS