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 Adolfo Bravo Ruiz, Pamela Romo Rodriguez, Alma Rosa Corrales Escobosa, Felix Gutierrez Corona, M. Isabel Roncero (Corresponding Author)

Research output: Contribution to journalArticle

3 Citations (Scopus)

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 ∆nit1∆nit2 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.
Original languageEnglish
Pages (from-to)493–507
Number of pages15
JournalCurrent Genetics
Volume64
Issue number2
Early online date17 Oct 2017
DOIs
Publication statusPublished - Apr 2018

Fingerprint

Pediculus
Fusarium
Nitrates
Virulence
Growth
Genes
Nitrate Reductase
Lycopersicon esculentum
Fungi
Nitrogen
Proteins
Chlorates
Aspergillus nidulans
Nitrites
Ammonium Compounds
Genome
Polymerase Chain Reaction

Keywords

  • Nitrogen metabolism
  • gene regulation
  • Plant pathogenicity
  • AreA

Cite this

Nitrate assimilation pathway (NAP) : role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity. / Gomez-Gil, Lucia; Camara Almiron, Jesus ; Rodriguez Carrillo, Patricia Lizett ; Medina, Cindy Nayely Olivares ; Bravo Ruiz, Gustavo Adolfo; Rodriguez, Pamela Romo ; Corrales Escobosa, Alma Rosa ; Corona, Felix Gutierrez ; Roncero, M. Isabel (Corresponding Author).

In: Current Genetics, Vol. 64, No. 2, 04.2018, p. 493–507.

Research output: Contribution to journalArticle

Gomez-Gil, L, Camara Almiron, J, Rodriguez Carrillo, PL, Medina, CNO, Bravo Ruiz, GA, Rodriguez, PR, Corrales Escobosa, AR, Corona, FG & Roncero, MI 2018, 'Nitrate assimilation pathway (NAP): role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity', Current Genetics, vol. 64, no. 2, pp. 493–507. https://doi.org/10.1007/s00294-017-0766-8
Gomez-Gil, Lucia ; Camara Almiron, Jesus ; Rodriguez Carrillo, Patricia Lizett ; Medina, Cindy Nayely Olivares ; Bravo Ruiz, Gustavo Adolfo ; Rodriguez, Pamela Romo ; Corrales Escobosa, Alma Rosa ; Corona, Felix Gutierrez ; Roncero, M. Isabel . / Nitrate assimilation pathway (NAP) : role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity. In: Current Genetics. 2018 ; Vol. 64, No. 2. pp. 493–507.
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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 ∆nit1∆nit2 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.",
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T2 - role of structural (nit) and transporter (ntr1) genes in Fusarium oxysporum f.sp. lycopersici growth and pathogenicity

AU - Gomez-Gil, Lucia

AU - Camara Almiron, Jesus

AU - Rodriguez Carrillo, Patricia Lizett

AU - Medina, Cindy Nayely Olivares

AU - Bravo Ruiz, Gustavo Adolfo

AU - Rodriguez, Pamela Romo

AU - Corrales Escobosa, Alma Rosa

AU - Corona, Felix Gutierrez

AU - Roncero, M. Isabel

N1 - 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.

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N2 - 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 ∆nit1∆nit2 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.

AB - 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 ∆nit1∆nit2 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.

KW - Nitrogen metabolism

KW - gene regulation

KW - Plant pathogenicity

KW - AreA

U2 - 10.1007/s00294-017-0766-8

DO - 10.1007/s00294-017-0766-8

M3 - Article

VL - 64

SP - 493

EP - 507

JO - Current Genetics

JF - Current Genetics

SN - 0172-8083

IS - 2

ER -