An efficient method for isolation of high-quality RNA from mycelium of toxigenic fungi Fusarium sp.

Мұқаба

Дәйексөз келтіру

Толық мәтін

Ашық рұқсат Ашық рұқсат
Рұқсат жабық Рұқсат берілді
Рұқсат жабық Тек жазылушылар үшін

Аннотация

Here, we describe a rapid and relatively cost-efficient method for the isolation and purification of RNA from mycelium of two species of plant-pathogenic fungi of Fusarium genus with different morphological and biochemical properties, F. graminearum and F. coffeatum. The method involves the use of guanidine hydrochloride-based buffer and spin columns from a commercial plasmid DNA extraction kit, and can be applied to both mycelia grown on nutrient agar media and liquid cultures of fungi. The yield of RNA isolated using the proposed protocol was 4–14 µg/100 mg of mycelium dry weight with RIN values up to 8.4. When optimizing the method, we propose to carry out a pre-lyophilization procedure, as well as the use of an RNAse inhibitor when isolating from cultures at late growth stages.

Толық мәтін

Рұқсат жабық

Авторлар туралы

А. Stakheev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Хат алмасуға жауапты Автор.
Email: stakheev.aa@gmail.com
Ресей, Moscow

D. Ryazantsev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Ресей, Moscow

N. Gabrielyan

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Ресей, Moscow

A. Poluboyarinova

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Ресей, Moscow

М. Taliansky

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Ресей, Moscow

S. Zavriev

Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences

Email: stakheev.aa@gmail.com
Ресей, Moscow

Әдебиет тізімі

  1. Fones H.N., Bebber D.P., Chaloner T.M., Kay W.T., Steinberg G., Gurr S.J. (2020) Threats to global food security from emerging fungal and oomycete crop pathogens. Nat. Food. 1(6), 332–342.
  2. Summerell B.A., Laurence M.H., Liew E.C.Y., Leslie J.F. (2010) Biogeography and phylogeography of Fusarium: a review. Fungal Divers. 44, 3–13.
  3. Ekwomadu T.I., Mwanza M. (2023) Fusarium fungi pathogens, identification, adverse effects, disease management, and global food security: a review of the latest research. Agriculture. 13(9), 1810.
  4. Kamle M., Mahato D.K., Gupta A., Pandhi S., Sharma B., Dhawan K., Vasundhara, Mishra S., Kumar M., Tripathi A.D., Rasane P., Selvakumar R., Kumar A., Gamlath S., Kumar P. (2022) Deoxynivalenol: an overview on occurrence, chemistry, biosynthesis, health effects and its detection, management and control strategies in food and feed. Microbiol. Res. 13, 292–314.
  5. Ropejko K., Twarużek M. (2021) Zearalenone and its metabolites – general overview, occurrence and toxicity. Toxins. 13, 35.
  6. Qu L., Wang L., Ji H., Fang Y., Lei P., Zhang X., Jin L., Sun D., Dong H. (2022) Toxic mechanism and biological detoxification of fumonisins. Toxins. 14, 182.
  7. Langevin F., Eudes F., Comaeu A. (2004) Effect of trichothecenes produced by Fusarium graminearum during Fusarium head blight development in six cereal samples. Eur. J. Plant Pathol. 110(7), 735–746.
  8. Merhej J., Richard-Forget F., Barreau C. (2011) Regulation of trichothecene biosynthesis in Fusarium: recent advances and new insights. Appl. Microbiol. Biotechnol. 91(3), 519–528.
  9. Woloshuk C.P., Shim W.-B. (2013) Aflatoxins, fumonisins and trichothecenes: a convergence of knowledge. FEMS Microbiol. Rev. 37(1), 94–109.
  10. Gil-Serna J., Vázquez C., Patiño B. (2020) Genetic regulation of aflatoxin, ochratoxin A, trichothecene, and fumonisin biosynthesis: a review. Int. Microbiol. 23(1), 89–96.
  11. Kolawole O., Meneely J., Petchkongkaew A., Elliott C. (2021) A review of mycotoxin biosynthetic pathways: associated genes and their expressions under the influence of climatic factors. Fungal Biol. Rev. 37(1), 8–26.
  12. Yang B., Wang Y., Tian M., Dai K., Zheng W., Liu Z., Yang S., Liu X., Shi D., Zhang H., Wang Y., Ye W., Wang Y. (2021) Fg12 ribonuclease secretion contributes to Fusarium graminearum virulence and induces plant cell death. J. Integr. Plant Biol. 63(2), 365–377.
  13. Qian H., Wang L., Wang B., Liang W. (2022) The secreted ribonuclease T2 protein FoRnt2 contributes to Fusarium oxysporum virulence. Mol. Plant Pathol. 23(9), 1346–1360.
  14. Ульянова В.В., Ваньков П.Ю., Зеленихин П.В., Шах Махмуд Р., Колпаков А.И., Ильинская О.Н. (2020) Секретируемые щелочные рибонуклеазы микромицетов. Ученые записки Казанского университета. Серия: естественные науки. 162(3), 335–349.
  15. Hansch C., McKarns S.C., Smith C.J., Doolitle D.J. (2000) Comparative QSAR evidence for a free-radical mechanism of phenol-induced toxicity. Chem. Biol. Interact. 127(1), 61–72.
  16. Sridar N., Krishnakishore C., Sandeep Y., Sriramnaveen P., Manjusha Y., Sivakumar V. (2011) Chloroform poisoning – a case report. Ren. Fail. 33(10), 1037–1039.
  17. Yaffe H., Buxdorf K., Shapira I., Ein-Gedi S., Moyal-Ben Zvi M., Fridman E., Moshelion M., Levi M. (2012) LogSpin: a simple, economical and fast method for RNA isolation from infected or healthy plants and other eukaryotic tissues. BMC Res. Notes. 5, 45.
  18. Минаева Л.П., Самохвалова Л.В., Завриев С.К., Стахеев А.А. (2022) Первое выявление гриба Fusarium coffeatum на территории Российской Федерации. Сельскохозяйственная биология. 57(1), 131–140.
  19. Farber J.M., Sanders G.W. (1986) Fusarin C production by North American isolates of Fusarium moniliforme. Appl. Environ. Microbiol. 51(2), 381–384.
  20. Stakheev A.A., Erokhin D.V., Meleshchuk E.A., Mikityuk O.D., Statsyuk N.V. (2022) Effect of compactin on the mycotoxin production and expression of related biosynthetic and regulatory genes in toxigenic Fusarium culmorum. Microorganisms. 10, 1347.
  21. Bustin S.A., Benes V., Garson J.A., Hellemans J., Huggett J., Kubista M., Mueller R., Nolan T., Pfaffl M.W., Shipley G.L., Vandesompele J., Witter C.T. (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR. Clin. Chem. 55(4), 611–622.
  22. Shcherbakova L., Mikityuk O., Arslanova L., Stakheev A., Erokhin D., Zavriev S., Dzhavakhiya V. (2021) Studying the ability of thymol to improve fungicidal effects of tebuconazole and difenoconazole against some plant pathogenic fungi in seed or foliar treatments. Front. Microbiol. 12, 629429.
  23. Sánchez-Rodríguez A., Portal O., Rojas L.E., Ocaña B., Mendoza M., Acosta M., Jiménez E., Höfte M. (2008) An efficient method of high-quality fungal total RNA to study the Mycosphaerella fijiensis-Musa spp. interaction. Mol. Biotechnol. 40(3), 299–305.
  24. Delira-Argumedo R., González-Mendoza D., Alarcón A. (2008) A rapid and versatile method for the isolation of total RNA from the filamentous fungus Trichoderma sp. Ann. Microbiol. 58(4), 761–763.
  25. Bernáldez V., Rodríguez A., Rodríguez M., Sánchez-Montero L., Córdoba J.J. (2017) Evaluation of different RNA extraction methods of filamentous fungi in various food matrices. LWT. 78(6), 47–53. https://doi.org/10.1016/j.lwt.2016.12.018
  26. Tu Q., Wang L., An Q., Shuai J., Xia X., Dong Y., Zhang X., Li G., He Y. (2023) Comparative transcriptomics identifies the key in planta-expressed genes Fusarium graminearum during infection of wheat varieties. Front. Genet. 14, 1166832.
  27. Miguel-Rojas C., Cavinder B., Townsend J.P., Trail F. (2023) Comparative transcriptomics of Fusarium graminearum and Magnaporthe oryzae spore germination leading up to infection. mBio. 14(1), e0244222.
  28. Zhang L., Zhou X., Li P., Wang Y., Hu Q., Shang Y., Chen Y., Zhu X., Feng H., Zhang C. (2023) Transcriptome profile of Fusarium graminearum treated by putrescine. J. Fungi. 9, 60.
  29. Schumann U., Smith N.A., Wang M.-B. (2013) A fast and efficient method for preparation of high-quality RNA from fungal mycelia. BMC Res. Notes. 6, 71.
  30. Shu C., Sun S., Chen J., Chen J., Zhou E. (2014) Comparison of different methods for total RNA extraction from sclerotia of Rhizoctonia solani. Electron. J. Biotechnol. 17(1), 50–54.
  31. Logemann J., Schell J., Willmitzer L. (1987) Improved method for the isolation of RNA from plant tissues. Anal. Biochem. 163(1), 16–20.
  32. Pearson G., Lago-Leston A., Valente M., Serrão E. (2006) Simple and rapid RNA extraction from freeze-dried tissue of brown algae and seagrasses. Eur. J. Phycol. 41(1), 97–104.
  33. García-Baldenegro C.V., Vargas-Arispuro I., Islas-Osuna M., Rivera-Domínguez M., Aispuro-Hernández E., Martínez-Téllez M.A. (2015) Total RNA quality of lyophilized and cryopreserved dormant grapevine buds. Electron. J. Biotechnol. 18, 50–54.
  34. Damsteegt E.L., McHugh N., Lokman P.M. (2016) Storage by lyophilization – resulting RNA quality is tissue dependent. Anal. Biochem. 511, 92–96.

Қосымша файлдар

Қосымша файлдар
Әрекет
1. JATS XML
2. Fig. 1. Results of the analysis of the integrity of RNA of F. graminearum (a) and F. coffeatum (b), performed on an Agilent 2100 Bioanalyzer (Agilent Technologies, USA). RNA was isolated from fungal mycelia on the 4th day of growth in a liquid nutrient medium.

Жүктеу (298KB)
3. Fig. 2. Electropherogram of RNA samples isolated from fungal mycelia on the 7th day of growth in liquid nutrient media. To the left of the marker (M) are samples isolated without the RiboCare RNase inhibitor, to the right – with the addition of the inhibitor. 1, 5 – F. graminearum, RNeasy Plant Mini Kit; 2, 6 – F. graminearum, method using 8 M guanidine hydrochloride; 3, 7 – F. coffeatum, RNeasy Plant Mini Kit; 4, 8 – F. coffeatum, method using 8 M guanidine hydrochloride.

Жүктеу (96KB)
4. Fig. 3. Graphs of the dependence of PCR threshold cycles on the degree of dilution of F. graminearum (a) and F. coffeatum (b) cDNA. The SD value is indicated next to each point.

Жүктеу (95KB)

© Russian Academy of Sciences, 2025