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Development of a rapid RNA extraction procedure from urediniospores of the leaf rust fungus, Puccinia triticina

Dutta, Summi, Kumar, Manish, Mukhopadhyay, Kunal
Journal of microbiological methods 2019 v.158 pp. 1-5
Puccinia recondita, RNA, cell walls, crushing, host-pathogen relationships, leaf rust, nitrogen, pathotypes, plant pathogenic fungi, plant tissues, ultrasonics, urediniospores
Obtaining high quality RNA in good quantities is often a requirement for plant-pathogen interaction studies, so it becomes very essential that a highly efficient method should be deployed to isolate RNA from minute quantities of fungal spores. The methods available to date, either require a high quantity of spores or the use of expensive chemicals. The protocol discussed here for RNA isolation from Puccinia triticina pathotype 77–5 urediniospores utilizes TRI Reagent as extraction buffer that is widely used for RNA isolation from plant tissues. Urediniospores have a tough cell wall as compared to other plant cells. Therefore, the protocol was optimized keeping the primary focus on quickly disrupting cell walls. Two different methods, one using a combination of liquid nitrogen and ultrasonic water-bath and the other method using micro-homogenizer were utilized for crushing the spores in the present study. The developed methods do not utilize mortar and pestle, instead they promote direct crushing of urediniospores in tubes; thereby minimizing sample loss and enhancing quality.