Identification of a Novel Small Cysteine-Rich Protein in the Fraction from the Biocontrol Fusarium oxysporum Strain CS-20 that Mitigates Fusarium Wilt Symptoms and Triggers Defense Responses in Tomato.

Larisa A Shcherbakova,Deborah R Fravel,Tatyana I Odintsova,Sergey K Zavriev,Alexander A Stakheev

doi:10.3389/fpls.2015.01207

Larisa A Shcherbakova, Deborah R Fravel + Show 3 more

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https://doi.org/10.3389/fpls.2015.01207

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Abstract

The biocontrol effect of the non-pathogenic Fusarium oxysporum strain CS-20 against the tomato wilt pathogen F. oxysporum f. sp. lycopersici (FOL) has been previously reported to be primarily plant-mediated. This study shows that CS-20 produces proteins, which elicit defense responses in tomato plants. Three protein-containing fractions were isolated from CS-20 biomass using size exclusion chromatography. Exposure of seedling roots to one of these fractions prior to inoculation with pathogenic FOL strains significantly reduced wilt severity. This fraction initiated an ion exchange response in cultured tomato cells resulting in a reversible alteration of extracellular pH; increased tomato chitinase activity, and induced systemic resistance by enhancing PR-1 expression in tomato leaves. Two other protein fractions were inactive in seedling protection. The main polypeptide (designated CS20EP), which was specifically present in the defense-inducing fraction and was not detected in inactive protein fractions, was identified. The nucleotide sequence encoding this protein was determined, and its complete amino acid sequence was deduced from direct Edman degradation (25 N-terminal amino acid residues) and DNA sequencing. The CS20EP was found to be a small basic cysteine-rich protein with a pI of 9.87 and 23.43% of hydrophobic amino acid residues. BLAST search in the NCBI database showed that the protein is new; however, it displays 48% sequence similarity with a hypothetical protein FGSG_10784 from F. graminearum strain PH-1. The contribution of CS20EP to elicitation of tomato defense responses resulting in wilt mitigating is discussed.

Highlights

The vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici (FOL) is one of the most destructive pathogens of greenhouse and field grown tomatoes (Benhamou et al, 1989; Fravel et al, 2003b; Anitha and Rabeeth, 2009; Kaur et al, 2010)
As with other microbial agents, nonpathogenic F. oxysporum strains employ several modes of action contributing to their biocontrol activity and the modes may vary depending on the strain or environment (Fravel et al, 2003b; Alabouvette et al, 2009)
Aliquots of 50–200 μl from the sterile ddH2O-dissolved lyophilized protein fraction V were added to cells, and changes in extracellular pH were monitored with time by using a universal pH-meter ZV-74 (ZIP, Russia) with an Orion semimicro pH electrode (Orion, USA) connected to a Cole Parmer recorder (USA)

Summary

Introduction

The vascular wilt pathogen Fusarium oxysporum f. sp. lycopersici (FOL) is one of the most destructive pathogens of greenhouse and field grown tomatoes (Benhamou et al, 1989; Fravel et al, 2003b; Anitha and Rabeeth, 2009; Kaur et al, 2010). Integrated protection against Fusarium wilt includes biological control as an important alternative or component of disease management This pathogen can be controlled by various microorganisms including nonpathogenic strains of F. oxysporum, which are used individually or together with soil bacteria (Fravel et al, 2003b; Silva and Bettiol, 2005; Alabouvette et al, 2009). Strain Fo47 controls FOL by priming of six genes involving in tomato defense responses (Aime et al, 2013) Another nonpathogenic F. oxysporum strain CS-20 does not affect the pathogen directly, but involves plantmediated mode of action and functions primarily by inducing the disease resistance (Larkin and Fravel, 1999; Panina et al, 2007)

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