Three Genes Related to Trehalose Metabolism Affect Sclerotial Development of Rhizoctonia solani AG-1 IA, Causal Agent of Rice Sheath Blight

doi:10.1016/j.rsci.2021.09.004

Abstract

Abstract:

Trehalose metabolism is related to the sclerotial development of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight (RSB). Here, we further elucidated the functions of three genes Rstre, Rstps1 and Rstpp that encode three key enzymes trehalase (TRE), alpha, alpha-trehalose- phosphate synthase (TPS1) and trehalose 6-phosphate phosphatase (TPP) in the sclerotial development of R. solani AG-1 IA. Due to the lack of a stable genetic transformation system for R. solani, the heterologous expression of these three genes in Pichia pastoris GS115 was performed. The results showed that reactive oxygen species (ROS) contents and enzyme activities in R. solani decreased significantly in the treatments of the fermentation broths of Rstps1 and Rstpp transformants, and that in the treatment of the fermentation broth of Rstre transformant visibly increased. Furthermore, the fermentation broths of the transformants of all the three genes were added to potato dextrose agar (PDA) medium for the cultivation of R. solani, as a result, the dry weight of sclerotia in each PDA plate containing the fermentation broths of Rstps1 and Rstpp transformants significantly increased compared with the control, and that of Rstre transformant obviously decreased. Finally, 178 proteins were found to interact with RSTPS1, and 16 of them were associated with ROS. Taken together, the findings suggest that all these three genes related to trehalose metabolism play important roles in the sclerotial development of R. solani AG-1 IA, and can be used as new targets for the development of novel high-efficiency fungicides for the controlling of RSB.

Key words: Rhizoctonia solani AG-1 IA, rice sheath blight, reactive oxygen species, trehalose metabolism, gene functional analysis, sclerotial development

Wang Chenjiaozi, Zhao Mei, Shu Canwei, Zhou Erxun. Three Genes Related to Trehalose Metabolism Affect Sclerotial Development of Rhizoctonia solani AG-1 IA, Causal Agent of Rice Sheath Blight[J]. Rice Science, 2022, 29(3): 268-276.

Figures/Tables 5

Fig. 1. Gene relative expression, Western blot and enzyme activities of Rstre, Rstps1 and Rstpp in different developmental stages of sclerotia in Rhizoctonia solani AG-1 IA. A, Relative expression levels of Rstre, Rstps1 and Rstpp at different sclerotial development stages. Samples were collected at seven different time points (36, 48, 60, 72, 120, 168 and 336 h of strain culture) during the sclerotial development. Data were normalized to transcripts encoding GAPDH, and shown as Mean ± SE (n = 3). B, Western blot of RSTPS1 and RSTPP at different sclerotial development stages. Anti- GAPDH was used as an internal control. C, Enzyme activities of RSTRE, RSTPS1 and RSTPP at different sclerotial development stages. Data are Mean ± SE (n = 3).

Fig. 2. Functional verification of Rstre (A), Rstps1 (B) and Rstpp (C) in Pichia pastoris GS115. pPIC9K-Rstre, pPIC9K-Rstps1 and pPIC9K-Rstpp are shown in red lines, and GS115 strain containing pPIC9K vector shown in blue line was used as a control. Solvent peak, glucose peak, trehalose peak and maltose peak are represented by blue, red, green and black arrows, respectively. nRIU is the refraction coefficient unit of differential refractive detector.

Fig. 3. Reactive oxygen species (ROS) contents in Pichia pastoris GS115 transformants of Rstre (A), Rstps1 (B) and Rstpp (C). ROS measurements were performed by the fluorescence assay. Mycelia were stained with 2’,7’-dichloro-dihydro-fluorescein diacetate for 20 min and visualized under a fluorescence microscope at 405 nm. Data are Mean ± SE (n = 3). * and ** indicate significant differences at P < 0.05 and P < 0.01 by the Duncan’s multiple range test.

Fig. 4. Effects of fermentation broths of Pichia pastoris GS115 transformants of Rstre, Rstps1 and Rstpp on the amounts of sclerotia. A?D, Morphology of sclerotia on potato dextrose agar plate containing GS115 fermentation broth (A), GS115-Rstre yeast fermentation broth (B), GS115-Rstps1 yeast fermentation broth (C) and GS115-Rstpp yeast fermentation broth (D). E, Effect of fermentation broth of yeast inverters on sclerotia of R. solani. Sclerotial di?erentiation (SDIF) was deﬁned as percent of dry weight of sclerotia (in respect to 100% SDIF of the control) per dish. Data are Mean ± SE (n = 3). * indicates significant difference at P < 0.05 by the Duncan's multiple range test.

Fig. 5. Gene Ontology (GO) annotation (A) and STRING assay (B) of newly discovered RSTPS1 interacting proteins.

References 30

[1]	Benaroudj N, Lee D H, Goldberg A L. 2001. Trehalose accumulation during cellular stress protects cells and cellular proteins from damage by oxygen radicals. J Biol Chem, 276: 24261-24267. PMID
[2]	Bookout A L, Cummins C L, Mangelsdorf D J, Pesola J M, Kramer M F. 2006. High-throughput real-time quantitative reverse transcription PCR. Curr Protoc Mol Biol, 15(8): 15.8. 1-15.8.21.
[3]	da Costa Morato Nery D, da Silva C G, Mariani D, Fernandes P N, Pereira M D, Panek A D, Eleutherio E C A. 2008. The role of trehalose and its transporter in protection against reactive oxygen species. Biochim Biophys Acta, 1780: 1408-1411.
[4]	Domingo D D, Bawingan P A, Bharathan S, Bharathan N. 2014. Molecular screening and characterization of dsRNA from wild- type and mutant strains of Rhizoctonia solani Kühn isolates. Philipp J Sci, 143: 61-72.
[5]	Fan K Q, Jin L Q, Zheng Y G. 2009. The enzymatic properties of trehalase and its exploitation as a target of new pesticides. Chem Bioeng, 26(4): 7-11.
[6]	Fang Y Z, Yang S, Wu G Y. 2002. Free radicals, antioxidants, and nutrition. Nutrition, 18: 872-879.
[7]	Feng S J, Shu C W, Wang C J Z, Jiang S F, Zhou E X. 2017. Survival of Rhizoctonia solani AG-1 IA, the causal agent of rice sheath blight, under different environmental conditions. J Phytopathol, 165(1): 44-52.
[8]	Georgiou C D, Tairis N, Sotiropoulou A. 2000. Hydroxyl radical scavengers inhibit sclerotial differentiation and growth in Sclerotinia sclerotiorum and Rhizoctonia solani. Mycol Res, 104: 1191-1196.
[9]	Georgiou C D, Patsoukis N, Papapostolou I, Zervoudakis G. 2006. Sclerotial metamorphosis in filamentous fungi is induced by oxidative stress. Integr Comp Biol, 46: 691-712.
[10]	Harlow E, Lane D. 1988. Antibodies:A Laboratory Manual. New York, USA: Cold Spring Harbor Laboratory Publishing.
[11]	Hottiger T, Schmutz P, Wiemken A. 1987. Heat-induced accumulation and futile cycling of trehalose in Saccharomyces cerevisiae. J Bacteriol, 169: 5518-5522. PMID
[12]	Jin K, Peng G X, Liu Y C, Xia Y X. 2015. The acid trehalase, ATM1, contributes to the in vivo growth and virulence of the entomopathogenic fungus, Metarhizium acridum. Fungal Genet Biol, 77: 61-67.
[13]	Lara-Ortíz T, Riveros-Rosas H, Aguirre J. 2003. Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans. Mol Microbiol, 50(4): 1241-1255. PMID
[14]	Leite F C B, da Rocha Leite D V, Pereira L F, de Barros Pita W, de Morais Junior M A. 2016. High intracellular trehalase activity prevents the storage of trehalose in the yeast Dekkera bruxellensis. Lett Appl Microbiol, 63: 210-214. PMID
[15]	Lu L, Shu C W, Liu C, Wang C J Z, Zhou E X. 2016. The impacts of natural antioxidants on sclerotial differentiation and development in Rhizoctonia solani AG-1 IA. Eur J Plant Pathol, 146(4): 729-740.
[16]	Luo Y, Li W M, Wang W. 2008. Trehalose: Protector of antioxidant enzymes or reactive oxygen species scavenger under heat stress? Environ Exp Bot, 63: 378-384.
[17]	Pan L, Zhang X Q, Wang J P, Ma X, Zhou M L, Huang L K, Nie G, Wang P X, Yang Z F, Li J. 2016. Transcriptional profiles of drought- related genes in modulating metabolic processes and antioxidant defenses in Lolium multiflorum. Front Plant Sci, 7: 519.
[18]	Pedreño Y, González-Párraga P, Martínez-Esparza M, Sentandreu R, Valentín E, Argüelles J C. 2007. Disruption of the Candida albicans ATC1 gene encoding a cell-linked acid trehalase decreases hypha formation and infectivity without affecting resistance to oxidative stress. Microbiology, 153: 1372-1381.
[19]	Richards A B, Krakowka S, Dexter L B, Schmid H, Wolterbeek A P M, Waalkens-Berendsen D H, Shigoyuki A, Kurimoto M. 2002. Trehalose: A review of properties, history of use and human tolerance, and results of multiple safety studies. Food Chem Toxicol, 40: 871-898. PMID
[20]	Shu C W, Sun S, Chen J L, Chen J Y, Zhou E X. 2014. Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani. Electron J Biotechnol, 17(1): 50-54.
[21]	Shu C W, Chen J L, Sun S, Zhang M L, Wang C J Z, Zhou E X. 2015. Two distinct classes of protein related to GTB and RRM are critical in the sclerotial metamorphosis process of Rhizoctonia solani AG-1 IA. Funct Integr Genomics, 15(4): 449-459.
[22]	Shu C W, Zhao M, Anderson J P, Garg G, Singh K B, Zheng W B, Wang C J Z, Yang M, Zhou E X. 2019. Transcriptome analysis reveals molecular mechanisms of sclerotial development in the rice sheath blight pathogen, Rhizoctonia solani AG1-IA. Funct Integr Genomics, 19: 743-758.
[23]	Suzuki S, Koga M, Niizeki N, Furuya A, Matsuo K, Tanahashi Y, Tsuchida E, Nohara F, Okamoto T, Nagaya K, Azuma H. 2013. Evaluation of glycated hemoglobin and fetal hemoglobin- adjusted HbA1c measurements in infants. Pediatr Diabetes, 14(4): 267-272.
[24]	Svanström A, Melin P. 2013. Intracellular trehalase activity is required for development, germination and heat-stress resistance of Aspergillus niger conidia. Res Microbiol, 164: 91-99. PMID
[25]	Theerakulpisut P, Gunnula W. 2012. Exogenous sorbitol and trehalose mitigated salt stress damage in salt-sensitive but not salt-tolerant rice seedlings. Asian J Crop Sci, 4: 165-170.
[26]	Tournu H, Fiori A, van Dijck P. 2013. Relevance of trehalose in pathogenicity: Some general rules, yet many exceptions. PLoS Pathog, 9(8): e1003447.
[27]	Wang C J Z, Pi L, Jiang S F, Yang M, Shu C W, Zhou E X. 2018. ROS and trehalose regulate sclerotial development in Rhizoctonia solani AG-1 IA. Fungal Biol, 122(5): 322-332.
[28]	Wibberg D, Jelonek L, Rupp O, Kröber M, Goesmann A, Grosch R, Pühler A, Schlüter A. 2014. Transcriptome analysis of the phytopathogenic fungus Rhizoctonia solani AG1-IB 7/3/14 applying high-throughput sequencing of expressed sequence tags (ESTs). Fungal Biol, 118: 800-813.
[29]	Xu L J, Zhao W, Cao C, Wang J, Wang Q, Wang Q G. 2015. Research progress of Jinggangmycin. Chin Agric Sci Bull, 31: 191-198. (in Chinese with English abstract)
[30]	Zou Y P, Ma L J, Dong H T, Tao F, Feng X J, Yuan D Z, Fan D S, Hu X P. 2017. PstTPS1, the trehalose-6-phosphate synthase gene of Puccinia striiformis f. sp. tritici, involves in cold stress response and hyphae development. Physiol Mol Plant Pathol, 100: 201-208.