Journal of Plant Physiology

Volume 190, 15 January 2016, Pages 79–94

Molecular biology

Suppression Subtractive Hybridization analysis provides new insights into the tomato (Solanum lycopersicum L.) response to the plant probiotic microorganism Trichoderma longibrachiatum MK1

  • a CNR, Institute of Biosciences and BioResources (IBBR), Research Division Portici, Via Università 133, 80055 Portici, NA, Italy
  • b Consiglio per la ricerca in agricoltura e l'analisi dell'economia agraria, Centro di ricerca per l'orticoltura, Via Cavalleggeri 25, 84098 Pontecagnano (SA), Italy
  • c Department of Ecological and Biological Sciences, University of Tuscia, Via S. Camillo De Lellis, 01100 Viterbo, Italy
  • d Plant-Microbe Interactions, Department of Biology, Faculty of Science, Utrecht University, Utrecht, Netherlands
  • e Department of Agricultural Sciences, University of Naples Federico II, via Università 100, 80055 Portici (NA), Italy
  • f CNR, Institute for Sustainable Plant Protection (IPSP), Via Università 133, 80055 Portici (NA), Italy
Received 28 July 2015, Revised 3 November 2015, Accepted 6 November 2015, Available online 17 November 2015

Abstract

Trichoderma species include widespread rhizosphere-colonising fungi that may establish an opportunistic interaction with the plant, resulting in growth promotion and/or increased tolerance to biotic and abiotic stresses. For this reason, Trichoderma-based formulations are largely used in agriculture to improve yield while reducing the application of agro-chemicals. By using the Suppression Subtractive Hybridization method, we identified molecular mechanisms activated during the in vitro interaction between tomato (Solanum lycopersicum L.) and the selected strain MK1 of Trichoderma longibrachiatum, and which may participate in the stimulation of plant growth and systemic resistance. Screening and sequence analysis of the subtractive library resulted in forty unique transcripts. Their annotation in functional categories revealed enrichment in cell defence/stress and primary metabolism categories, while secondary metabolism and transport were less represented. Increased transcription of genes involved in defence, cell wall reinforcement and signalling of reactive oxygen species suggests that improved plant pathogen resistance induced by T. longibrachiatum MK1 in tomato may occur through stimulation of the above mechanisms. The array of activated defence-related genes indicates that different signalling pathways, beside the jasmonate/ethylene-dependent one, collaborate to fine-tune the plant response. Our results also suggest that the growth stimulation effect of MK1 on tomato may involve a set of genes controlling protein synthesis and turnover as well as energy metabolism and photosynthesis. Transcriptional profiling of several defence-related genes at different time points of the tomato–Trichoderma interaction, and after subsequent inoculation with the pathogen Botrytis cinerea, provided novel information on genes that may specifically modulate the tomato response to T. longibrachiatum, B. cinerea or both.

Abbreviations

  • BCA, biocontrol agent;
  • ET, ethylene;
  • DPI, days post inoculation;
  • ISR, induced systemic resistance;
  • LHC, light-harvesting complex;
  • MAMPS, Microbe-Associated Molecular Patterns;
  • PGP, plant growth promotion;
  • PGPF, plant growth promoting fungi;
  • PGPR, plant growth promoting rhizobacteria;
  • SAR, Systemic Acquired Resistance;
  • SGN, SOL Genomics Network;
  • SSH, Suppression Subtractive Hybridization

Keywords

  • Induced systemic resistance;
  • Plant growth promotion;
  • Differential cDNA library;
  • Transcriptome analysis;
  • Rhizosphere microbiome;
  • Plant pathogens

1. Introduction

Soil-borne beneficial microbes, like plant growth promoting rhizobacteria (PGPR) or fungi (PGPF), rhizobia, and mycorrhizal fungi, are well-known plant stimulators and can protect plants from abiotic and biotic stresses (Pozo and Aguilar, 2007, Bonfante and Genre, 2010, Berendsen et al., 2012, Caporale et al., 2014, Ruocco et al., 2015 and Vos et al., 2015).

The direct and indirect biocontrol activity of rhizosphere-competent fungi of the genus Trichoderma is widely recognized ( Harman et al., 2004 and Shoresh et al., 2010). Several strains are also able to stimulate plant growth (Lorito and Woo, 2015), improve nutrient uptake (Zhao et al., 2014), and contribute to plant hormonal balance and volatile production ( Harman et al., 2004, Shoresh et al., 2005, Battaglia et al., 2013 and Ruocco et al., 2015). It has also been demonstrated that these PGPF can protect plants from abiotic stresses and affect their direct and indirect resistance to insect pests ( Bae et al., 2009, Mastouri et al., 2010, Mastouri et al., 2012, Battaglia et al., 2013, Brotman et al., 2013 and Caporale et al., 2014). Thanks to their proved efficacy, a number of Trichoderma strains have been selected for application in agriculture, with a few hundreds of formulations registered worldwide, while several strains have been deeply studied at the laboratory level for their peculiar biological and genetic features ( Lorito and Woo, 2015).

The molecular mechanisms that regulate direct Trichoderma biocontrol activity have been ascertained ( Atanasova et al., 2013), those enabling Trichoderma strains to promote indirect defence against pathogens and pests and, even more so, plant growth have not been fully uncovered. Several reports indicate that the ability of Trichoderma spp. to activate induced systemic resistance (ISR) against pathogen infections is mediated by jasmonate (JA)- and ethylene (ET)-dependent mechanisms and requires transient expression of defence genes ( Shoresh et al., 2005 and Korolev et al., 2008). Moreover, long-lasting up-regulation of salicylic acid (SA)-responsive genes was demonstrated in tomato interacting with Trichoderma harzianum T22, whose modulation, together with JA-induced gene expression, contributed to increased resistance to the pathogen Botrytis cinerea ( Tucci et al., 2011). Recently, increasing evidence is accumulating that both JA/ET and SA signalling may be triggered by Trichoderma in crop and model plants ( Segarra et al., 2007, Tucci et al., 2011, Mathys et al., 2012, Perazzolli et al., 2012 and Martinez-Medina et al., 2013). Only a few studies have addressed the molecular mechanisms underlying the promotion of plant growth by Trichoderma species. Auxin signalling was demonstrated to be important for biomass production induced by Trichoderma virens ( Contreras-Cornejo et al., 2009) and increased transcription of IAA-related genes was observed in Arabidopsis thaliana roots after T. harzianum inoculation ( Brotman et al., 2013). Moreover, proteomic approaches indicated increased photosynthesis and carbohydrate metabolism in Trichoderma-treated plants ( Segarra et al., 2007 and Shoresh and Harman, 2008), which were suggested to be related to enhanced growth response.