Expression of MfAvr4 in banana leaf sections with black leaf streak disease caused by Mycosphaerella fijiensis: a technical validation

Published Date

pp 1-8

First online: 06 August 2016

Title 

Expression of MfAvr4 in banana leaf sections with black leaf streak disease caused by Mycosphaerella fijiensis: a technical validation

• Author 

• Cecilia Mónica Rodríguez-García 
• , Abril Diane Canché-Gómez
• , Luis Sáenz-Carbonell
• , Leticia Peraza-Echeverría
• , Blondy Canto-Canché
• , Ignacio Islas-Flores
• , Santy Peraza-Echeverría

Abstract

The objective of this study was to validate the use of banana leaf sections as a technique to study the molecular interaction between Mycosphaerella fijiensis and Musaspp. without the interference of biotic and abiotic factors that commonly occur under field conditions. The growth of M. fijiensis in banana leaf sections was evaluated and compared with the growth of the fungus in leaves under field conditions. Growth comparison was carried out through the absolute quantification by real-time PCR of a segment of the β-tubulin gene of M. fijiensis. Validation of the banana leaf sections technique consisted in monitoring M. fijiensis MfAvr4 gene expression and its relative quantification by real-time PCR in banana leaf sections. With this technique, it was shown that the growth of M. fijiensis and MfAvr4 gene expression were similar to those observed in infected leaves in the field. These quantitative real-time PCR results support the suitability of using banana leaf sections for molecular studies of gene expression in M. fijiensis-Musa spp. interactions.

Supplementary Material

13313_2016_431_Fig5_ESM.gif (14629)

Figure SM1 Standard curve regression between the threshold cycle (Ct) values for the β-tubulin gene and the log10 of M. fijiensis DNA. Serial dilutions (1:10) of gDNA (100 ng/ μL) from mycelia of M. fijiensis were prepared to construct the standard curve. Black circles represent the mean of data used to calculate the PCR efficiency value using the formula: 10(−1/ slope) − 1 (GIF 14 kb)

Blah13313_2016_431_MOESM1_ESM.tif (1561040)

High resolution image (TIFF 1524 kb)

Blah13313_2016_431_Fig6_ESM.gif (22344)

Figure SM2 Schematic representation of the validation of inter-assay using a single sample naturally infected (stage 5) on different days. Five replicates of the same DNA sample were analysed in five repeated experiments; different letters indicate statistically significant differences (P < 0.05) determined by ANOVA and Fisher tests (GIF 21 kb)

Blah13313_2016_431_MOESM2_ESM.tif (2888676)

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Blah13313_2016_431_Fig7_ESM.gif (13363)

Figure SM3 Schematic representation of the validation of inter-sample using samples naturally infected (stage 3). Three different gDNA samples of these banana leaves were analysed in a single run. Different letters indicate statistically significant differences (P < 0.05) determined by ANOVA and Fisher tests (GIF 13 kb)

Blah13313_2016_431_MOESM3_ESM.tif (2035284)

High resolution image (TIFF 1987 kb)

Blah13313_2016_431_Fig8_ESM.gif (25304)

Figure SM4 Standard curve regression between the threshold cycle (Ct) values for the β-tubulin (a) and MfAvr4 (b) genes and the log10 of M. fijiensis, to validate the relative quantification in artificially infected banana leaf sections, and in naturally infected banana leaves. Serial dilutions (1:10) of cDNA (200 ng/μL) from mycelia of M. fijiensiswere prepared to construct the standard curve. Black circles represent the mean of data used to calculate the PCR efficiency value using the formula 10(−1/ slope) − 1 (GIF 24 kb)

Blah13313_2016_431_MOESM4_ESM.tif (2155340)

High resolution image (TIFF 2104 kb)

Blah13313_2016_431_MOESM5_ESM.docx (54208)

ESM 1 (DOCX 115 kb)

References

1. Abadie C, Zapater MF, Pignolet L, Carlier J, Mourichon X (2008) Artificial inoculation on plants and banana leaf pieces with Mycosphaerella spp., Responsible for Sigatoka leaf spot diseases. Fruits 63(5):319–323
2. Arzanlou M, Abeln E, Kema G, Waalwijk C, Carlier J, Vries de I, et al. (2007) Molecular diagnostics for the Sigatoka disease complex of banana. Phytopathology 97(9):1112–1118
3. Balint-Kurti PJ, May GD, Churchill ACL (2001) Development of a transformation system for Mycosphaerellapathogens of banana: a tool for the study of host/pathogen interactions. FEMS Microbiol Lett 195(1):9–15
4. Beveraggi A, Mourichon X, Salle G (1995) Étude comparée des premières étapes de l’infection chez des bananiers sensibles et résistants infectés par le Cercospora fijiensis (Mycosphaerella fijiensis) Agent responsable de la maladie des raies noires. Can J Bot 73(9):1328–1337
5. Brooks FE (2008) Detached-leaf bioassay for evaluating Taro resistance to Phytophthora colocasiae. Plant Dis 92(1):126–131CrossRef
6. Cho Y, Hou S, Zhong S (2008) Analysis of expressed sequence tags from the fungal banana pathogen Mycosphaerella fijiensis. The Open Mycology Journal 2:61–73CrossRef
7. Churchill ACL (2011) Mycosphaerella fijiensis, the black leaf streak pathogen of banana: progress towards understanding pathogen biology and detection, disease development, and the challenges of control. Mol Plant Pathol 12(4):307–328CrossRefPubMed
8. Couoh-Uicab Y, Islas-Flores I, Kantún-Moreno N, Zwiers LH, Tzec-Simá M, Peraza-Echeverría S, et al. (2012) Cloning, in silico structural characterization and expression analysis of MfAtr4, an ABC transporter from the banana pathogen Mycosphaerella fijiensis. Afr J Biotechnol 11(1):54–79
9. D’Hont A, Denoeud F, Aury JM, Baurens FC, Carreel F, Garsmeur O, et al. (2012) The banana (Musa acuminata) genome and the evolution of monocotyledonous plants. Nature 488(7410):213–217
10. De Lapeyre de Bellaire L, Fouré E, Abadie C, Carlier J (2010) Black Leaf Streak Disease is challenging the banana industry. Fruits 65(6):327–342CrossRef
11. Department of Energy Joint Genome Institute (2010) Mycosphaerella fijiensis v2.0 [http://​genomeportal.​jgi.​doe.​gov/​Mycfi2/​Mycfi2.​home.​html]. Accessed Jul 2013
12. Fouré E (1985) Black leaf streak disease of bananas and plantains (Mycosphaerella fijiensis Morelet). Study of the symptoms and stages of the disease in Gabon. IRFA-CIRAD, Paris
13. Guo JR, Schnieder F, Verreet JA (2006) Presymptomatic and quantitative detection of Mycosphaerella graminicoladevelopment in wheat using a real-time PCR assay. FEMS Microbiol Lett 262(2):223–229CrossRefPubMed
14. Hamid R, Khan MA, Ahmad M, Ahmad MM, Abdin MZ, Musarrat J, Javed S (2013) Chitinases: An update. Int J Pharm. Bio. Sci 5(1):21–29 [doi:10.​4103/​0975-7406.​106559]. Accesed Feb 2016
15. Hoss L, Helbig J, Bochow H (2000) Function of host and fungal metabolites in resistance response of banana and plantain in the black Sigatoka disease pathosystem (Musa spp. Mycosphaerella fijiensis). Phytopathol 148(7–8):387–394CrossRef
16. Islas-Flores I, Peraza-Echeverría L, Canto-Canché B, Rodríguez-García CM (2006) Extraction of high-quality, melanin-free RNA from Mycosphaerella fijiensis for cDNA preparation. Mol Biotechnol 34(1):45–50CrossRefPubMed
17. Johanson A (1995) Detection of banana leaf spot pathogens by PCR. Bulletin, Organisation Européene et Méditerranéene pour la Protection des Plantes/European and Mediterranean Plant Protection Organization (OEP/EPPO) 25(1–2):99–107
18. Kantún-Moreno N, Vázquez-Euán R, Tzec-Simá M, Peraza-Echeverría L, Grijalva-Arango R, Rodríguez-García C, James AC, Ramírez-Prado J, Islas-Flores I, Canto-Canché B (2013) Genome-wide in silico identification of GPI proteins in Mycosphaerella fijiensis and transcriptional analysis of two GPI-anchored b-1,3-glucanosyltransferases. Mycologia 105(2):285–296CrossRefPubMed
19. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25(4):402–408CrossRefPubMed
20. Marín DH, Romero RA, Guzmán M, Turner B (2003) Black Sigatoka: An Increasing Threat to Banana Cultivation. Plant Dis 87(3):208–222CrossRef
21. Marshall R, Kombrink A, Motteram J, Loza-Reyes E, Lucas J, Hammond-Kosack KE, et al. (2011) Analysis of two in planta expressed LysM effector homologs from the fungus Mycosphaerella graminicola reveals novel functional properties and varying contributions to virulence on wheat. Plant Physiol 156(2):756–769CrossRefPubMedPubMedCentral
22. Mendoza-Rodríguez M, Sánchez-Rodríguez A, Acosta-Suárez M, Berkis R, Portal O, Jiménez E (2006) Construcción y secuenciación parcial de una biblioteca sustractiva en ‘Calcutta 4’ (Musa AA) en estadio temprano de infección con Mycosphaerella fijiensis Morelet. Biotecnología Vegetal 6(4):213–217
23. Motteram J, Küfner I, Deller S, Brunner F, Hammond-Kosack KE, Nürnberger T, et al. (2009) Molecular characterization and functional analysis of MgNLP, the sole NPP1 domain-containing protein, from the fungal wheat leaf pathogen Mycosphaerella graminicola. Mol Plant Microbe In 22(7):790–799CrossRef
24. Peraza-Echeverría L, Rodríguez-García CM, Zapata-Salazar DM (2008) A rapid, effective method for profuse in vitro conidial production of Mycosphaerella fijiensis. Australas Plant Path 37(5):460–463CrossRef
25. Portal O, Izquierdo Y, Vleesschauwer DD, Sánchez-Rodríguez A, Mendoza-Rodríguez M, Acosta-Suárez M, Ocaña B, Jiménez E, Höfte M (2011) Analysis of expressed sequence tags derived from a compatible Mycosphaerella fijiensis- banana interaction. Plant Cell Rep 30(5):913–928CrossRefPubMed
26. Qi M, Yang Y (2002) Quantification of Magnaporthe grisea during infection of rice plants using real-time polymerase chain reaction and Northern blot/phosphoimaging analysis. Phytopathology 92(8):870–876CrossRefPubMed
27. Radonic A, Thulke S, Mackay MI, Landt O, Siegert W, Nitsche A (2004) Guideline to reference gene selection for quantitative real-time PCR. Biochem Bioph Res Co 313(4):856–862CrossRef
28. Raymaekers M, Smets R, Maes B, Cartuyvels R (2009) Checklist for optimization and validation of real-time PCR assays. J Clin Lab Anal 23(3):145–151CrossRefPubMed
29. Rodríguez-García CM, Peraza-Echeverria L, Islas-Flores IR, Canto-Canché BB, Grijalva-Arango R (2010) Isolation of retro-transcribed RNA from in vitro Mycosphaerella fijiensis infected banana leaves. Genet Mol Res 9(3):1460–1468CrossRefPubMed
30. Sánchez-García C, Cruz-Martín M, Alvaro-Copó Y, Roja L, Leiva-Mora M, Acosta-Suaréz M, Roque B (2012) Detección y cuantificación de quitinasas en hojas de banano (Musa spp) inoculadas con Mycosphaerella fijiensis. Biotecnología Vegetal 12(2):119–124
31. Sánchez-Rodríguez YA (2012) Cuantificación por PCR en tiempo real de la biomasa de Mycosphaerella fijiensis en plantaciones de banano de Teapa, Tabasco. Master’s Thesis. CICY, Mérida, Yucatán, México
32. Stergiopoulos I, van den Burg HA, Ökmen B, Beenen HG, van Liere S, Kema HJG, et al. (2010) Tomato Cf resistance proteins mediate recognition of cognate homologous effectors from fungi pathogenic on dicots and monocots. P Natl Aca Sci USA 107(16):7610–7615CrossRef
33. Twizeyimana M, Ojiambo PS, Tenkouano A, Ikotun T, Bandyopadhyay R (2007) Rapid screening of Musa species for resistance to Black Leaf Streak using in vitro plantlets in tubes and detached leaves. Plant Dis 91(3):308–314CrossRef
34. Van der Does CH, Duyvesteijn RGE, Goltstein PM, van Schie CCN, Manders EMM, Cornelissen BJC, et al. (2008) Expression of effector gene SIX1 of Fusarium oxysporum requires living plant cells. Fungal Genet Biol 45(9):1257–1264CrossRefPubMed
35. Vleeshouwers GAA, van Dooijeweert W, Keizer LCP, Sijpkes L, Govers F, Colon LT (1999) A laboratory assay for Phytophthora infestans resistance in various Solanum species reflects the field situation. Eur J Plant Pathol 105(3):241–250CrossRef
36. Weising K, Kaemmer D, Epplen JT, Weigand F, Saxena M, Kahl G (1991) DNA fingerprinting of Ascochyta rabieiwith synthetic oligodeoxynucleotides. Curr Genet 19(6):483–489CrossRef
37. Winton LM, Stone JK, Watrud LS, Hansen EM (2002) Simultaneous one-tube quantification of host and pathogen DNA with Real-Time Polymerase Chain Reaction. Phytopathology 92(1):112–116CrossRefPubMed
38. Yessad S, Manceau C, Luisetti J (1992) A detached leaf assay to evaluate virulence and pathogenicity of strains of Pseudomonas syringae pv. syringae on pear. Plant Dis 76(4):370–373CrossRef

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