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Current Genetics

, Volume 64, Issue 2, pp 493–507 | Cite as

Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity

  • Lucia Gomez-Gil
  • Jesus Camara Almiron
  • Patricia Lizett Rodriguez Carrillo
  • Cindy Nayely Olivares Medina
  • Gustavo Bravo Ruiz
  • Pamela Romo Rodriguez
  • Alma Rosa Corrales Escobosa
  • Felix Gutierrez Corona
  • M. Isabel RonceroEmail author
Original Article

Abstract

The tomato pathogen Fusarium oxysporum f.sp. lycopersici possesses the capability to use nitrate as the only nitrogen source under aerobic and anaerobic conditions and to activate virulence-related functions when cultivated in the presence of nitrate, but not in ammonium. The genome of F. oxysporum f.sp. lycopersici encodes three paralogs nitrate reductase (NR) genes (nit1, nit2 and nit3) and one predicted ortholog of the Aspergillus nidulans high-affinity nitrate/nitrite transporters NtrA and NtrB, named ntr1. We set out to clarify the role of nit1, nit2, nit3 and ntr1 genes in nitrate assimilation and in the virulence of F. oxysporum f.sp. lycopersici. Quantitative RT-PCR analysis revealed that only nit1, nit2 and ntr1 are expressed at significant levels during growth in nitrate as the only nitrogen source. Targeted deletion of nit1 and ntr1, but not of nit2 or nit3, severely impaired growth of F. oxysporum on nitrate as nitrogen source, indicating that Nit1 and Ntr1 proteins are involved in nitrate assimilation by the fungus; biochemical analysis of nit mutants indicated that Nit1 and Nit2 enzymes contribute to about 50 and 30% of the total NR activity, respectively. In addition, a spontaneous chlorate-resistant mutant derived from F. oxysporum 4287, denoted NitFG, was characterized, showing inability to grow in nitrate under aerobic and anaerobic conditions and low levels of NR activity, in spite of its increased transcription levels of nit1 and nit2 genes. Tomato plant infection assays showed that NitFG and ∆ntr1 mutants induced an earlier death in tomato plants, whereas the single mutants ∆nit1, ∆nit2 and ∆nit1nit2 double mutant showed a mortality rate similar to the wild-type strain. Taken together, these results indicate that the Nit1 and Ntr1 proteins are important for nitrate assimilation of F. oxysporum f.sp. lycopersici incubated under aerobic and anaerobic conditions and that this metabolic process is not essential for the virulence of the fungus. These observations open new questions about the role of Nit1, Nit2, and Nit3 proteins in other routes of nitrate metabolism in this pathogenic fungus and in the possible regulatory role that can be exerted by the AreA protein in these routes.

Keywords

Nitrogen metabolism Gene regulation Plant pathogenicity AreA 

Notes

Acknowledgements

The authors gratefully acknowledge Esther Martinez Aguilera for valuable technical assistance. They thank Prof. Jose Manuel Siverio (University of La Laguna, Tenerife, Spain) for providing the antibody directed against the enzyme NR of Hansenula polymorpha, and Prof. Antonio Di Pietro (University of Cordoba, Spain) for his advise and discussions. This research was supported by Junta de Andalucía (Grant CVI-7319), the Spanish Ministerio de Economia y Competitividad (Grants BIO2013-47870 and BIO2016-78923-R). G.B.R. had a postdoctoral position from Consejeria de Economia, Innovacion, Ciencia y Empresa, Junta de Andalucía. P.L.R.C. received a fellowship from CONACyT, Mexico.

Supplementary material

294_2017_766_MOESM1_ESM.pdf (571 kb)
Suppl. Fig. 1.- Deletion of nit1. (PDF 571 KB)
294_2017_766_MOESM2_ESM.pdf (335 kb)
Suppl. Fig. 2.- Deletion of nit2. (PDF 335 KB)
294_2017_766_MOESM3_ESM.pdf (327 kb)
Suppl. Fig. 3.- Deletion of nit3. (PDF 326 KB)
294_2017_766_MOESM4_ESM.pdf (568 kb)
Suppl. Fig. 4.- Deletion of ntr1 (PDF 567 KB)
294_2017_766_MOESM5_ESM.pdf (371 kb)
Suppl. Fig. 5.- Construction of Δnit1Δnit2 double mutant. (PDF 371 KB)
294_2017_766_MOESM6_ESM.pdf (689 kb)
Suppl. Fig. 6.- Complementation of the NitFG mutant by co-transformation with the areA + wild-type allele and the HygR cassette. A Physical structure of the areA genomic region. Positions of restriction sites EcoR I are shown. Black (forward) and red (reverse) arrowheads indicate the position of primers used for amplification of each fragment. Scale bar indicates 1kb. B Southern analyses of EcoR I digested gDNA from HygR transformants using the indicated probe. (PDF 688 KB)
294_2017_766_MOESM7_ESM.pdf (1.5 mb)
Suppl. Fig. 7.- Phylogenetic relationships among 12 NR deduced proteins from F. oxysporum f.sp. lycopersici FOXG_04181 (Nit1), FOXG_03770 (Nit2), FOXG_02795 (Nit3), F. verticillioides (FVEG_07298, FVEG_12090, FVEG_01627), F. graminearum, (FGSG_01947), F. fujikuroi (FFUJ_06561, FFUJ_12277, FFUJ_14513) and A. nidulans (ANID_1006; ANIA_08449). A, The PHYML 3.0 program (Guindon and Gascuel 2003) was used to perform a 1,000 nonparametric bootstrap phylogenetic analysis of the resulting alignment of 212 amino acid characters with the maximum likelihood method after optimization of the settings by the MODELGENERATOR program, version 0.85 (Keane et al. 2006). B, Sequence alignment used the ClustalW method with the Bioedit 7.0.0 program (Hall 1999). Identical and similar amino acids are highlighted on a light and dark grey background, respectively. (PDF 1488 KB)

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Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Lucia Gomez-Gil
    • 1
  • Jesus Camara Almiron
    • 1
  • Patricia Lizett Rodriguez Carrillo
    • 2
  • Cindy Nayely Olivares Medina
    • 2
  • Gustavo Bravo Ruiz
    • 1
  • Pamela Romo Rodriguez
    • 2
  • Alma Rosa Corrales Escobosa
    • 3
  • Felix Gutierrez Corona
    • 2
  • M. Isabel Roncero
    • 1
    Email author
  1. 1.Departamento de GeneticaUniversidad de Cordoba and Campus de Excelencia Agroalimentario (ceiA3)CordobaSpain
  2. 2.Departamento de Biologia y, DCNEUniversidad de GuanajuatoGuanajuatoMexico
  3. 3.Departamento de Quimica, DCNEUniversidad de GuanajuatoGuanajuatoMexico

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