The role of abscisic acid in the defence response of tomato (Solanum lycopersicum) to the necrotrophic pathogens Botrytis cinerea and Erwinia chrysanthemi

In order to cope with the constant threat of a wide range of potentially harmful micro-organisms, plants have developed an impressive constitutive and inducible defensive machinery of enormous complexity to combat pathogen invasion. Plant hormones are not only important for controlling plant development, but are also essential to regulate plant responses to the environment. The plant hormones salicylic acid (SA), jasmonate (JA) and ethylene (ET) are classically associated with plant pathogen defence responses as their increase in concentrations upon pathogen recognition, which leads to the activation of specific signalling cascades and pathogen defence gene expression, is important for resistance. In contrast, the plant hormone abscisic acid (ABA) has a well-established function in activating plant responses to abiotic stresses (such as cold, drought and salinity) by regulating stomatal aperture and by activating stress-responsive genes, but the effect of ABA on plant-pathogen interactions has long been a neglected research topic. Although exceptions exist, basal or high ABA levels are in most cases associated with susceptibility, while reduction of plant ABA levels often leads to increased resistance. However, our knowledge on the mechanisms of ABA-induced susceptibility is still very scarce and fragmentary. In the present work, we have explored the influence of ABA on the defence responses of tomato towards biotic stress. We have shown that ABA deficiency in the sitiens tomato mutant results in increased resistance towards the necrotrophic fungus Botrytis cinerea and the necrotrophic bacterium Erwinia chrysanthemi, two pathogens for which the occurrence of resistance is very rare. Comparison of gene expression in sitiens and wild-type tomato with TOM1 microarrays revealed that defence-related transcript accumulation prior to infection is higher in sitiens than in wild type. Moreover, further elevation of defence gene expression after pathogen attack is also stronger in sitiens, both in number of genes and their expression levels. These results show that ABA-deficiency results in priming for pathogen defence. Annotation of the genes differentially regulated between in sitiens and wild type showed elevated expression levels in sitiens of genes encoding SA-inducible PR proteins and of genes involved in SA biosynthesis, which confirmed earlier findings that ABA deficiency leads to hyperinduction of SA-dependent defence responses. Thorough analysis of the defence reactions that were hyperactivated in sitiens upon inoculation with B. cinerea and E. chrysanthemi revealed an essential role for hydrogen peroxide accumulation in the defence response towards both pathogens. Compared to wild-type tomato, hydrogen peroxide accumulation was earlier and more extensive in sitiens. The necessity of this defence reaction in the establishment of resistance in sitiens was demonstrated by disruption of the early and strong hydrogen peroxide accumulation. Removal of hydrogen peroxide with the antioxidants catalase and ascorbate and blocking hydrogen peroxide production with diphenilene iodonium increased the levels of susceptibility in sitiens. Accumulation of reactive oxygen species (ROS), such as hydrogen peroxide, during pathogen attack is a well-known phenomenon and its role in arresting biotrophic pathogens is firmly established. However, ROS accumulation is supposed to have a negative effect on defence against necrotrophic pathogens such as B. cinerea, since elevation of in planta ROS levels sets of a hypersensitive response, leading to increased plant tissue colonisation by necrotrophic pathogens. Our results clearly oppose the generally accepted theorem that plant defence-related ROS formation aids necrotrophs in their pathogenicity, and show that a timely hyperproduction of hydrogen peroxide is efficient in protecting the sitiens tomato mutant against necrotrophic pathogen attack. Although ROS can have other roles in defence, such as functioning as a signalling intermediate, having a direct antimicrobial effect or leading to a hypersensitive response, we have demonstrated that hyperaccumulation of hydrogen peroxide in sitiens leads to cell wall fortification, which results in containment of E. chrysanthemi and B. cinerea. Hydrogen peroxide accumulation in sitiens was accompanied by earlier and increased activation of extracellular peroxidases and a strong histochemical-detected cell wall fortification at the site of pathogen attack. These findings were consistent with the immediate hydrogen peroxide-fuelled peroxidative cross-linking of structural cell wall proteins and peroxidative incorporation of phenolic compounds during an oxidative burst. Furthermore, analysis of the genes activated in sitiens during B. cinerea inoculation revealed an overrepresentation of genes involved in cell wall modification. Microscopical analysis of pathogen progress in sitiens leaf tissue showed that both E. chrysanthemi (visualised directly as intercellular micro-colonies) and B. cinerea (visualised indirectly as progress of pectin degradation) were arrested by hydrogen peroxide-induced cell wall fortification. The site of pathogen arrest, i.e. the site of cell wall fortification, differed for E. chrysanthemi and B. cinerea, which was at least partly due to the nature of each inoculation procedure. B. cinerea penetrates the adaxial leaf cuticula and outer epidermal cell wall and was blocked by wall fortifications of the anticlinal epidermal sitiens cells. The arrest of E. chrysanthemi, which is infiltrated in the leaf tissue, was located at the sites of sitiens cell wall fortification at the border of the infiltration zone. Deposition of callose at the site of pathogen entry was previously demonstrated to be influenced by ABA. We have shown that callose deposition after B. cinerea inoculation is weaker in sitiens compared to the wild type. Inhibition of callose synthesis with 2-deoxy-D-glucose did not affect resistance in sitiens, but caused additional susceptibility in wild type. These findings indicate that callose deposition is not part of sitiens defence responses that are effective in blocking B. cinerea and suggest that callose deposition only contributes to wild-type tomato basal resistance. There are strong indications that the rapid and powerful defence responses in sitiens are triggered by recognition of endogenous plant cell wall elicitors. Both E. chrysanthemi and B. cinerea trigger the same type of defence responses in sitiens, which conflicts with the involvement of pathogen-specific elicitors. In addition, both pathogens produce great amounts of cell wall-degrading enzymes (CWDEs) and rely largely on cell wall pectin decomposition for their virulence. We have also shown that type II secretion-negative E. chrysanthemi mutants, which are incapable of secreting CWDEs, fail to fully activate sitiens defence responses. Furthermore, defence responses are elicited by bacteria-free CDWE-containing E. chrysanthemi culture filtrate. Since we found no differences between sitiens and wild-type in the capacity to resist E. chrysanthemi cell wall degradation, we have hypothesised that changes in sitiens cell wall composition could be responsible for differences in the release of cell wall oligomers, which are known and potent elicitors of defence responses upon pathogenic cell wall degradation. Alternatively, enhanced sensing of these oligomers in sitiens could also be responsible for the faster activation of defence. We found support for both hypotheses, as immunolocalisation of different cell wall components demonstrated differences between sitiens and wild-type in pectin distribution and in the presence of arabinogalactan proteins, which are presumably involved in signalling at the cell surface. Further experimental data will be needed expand our knowledge sitiens defence elicitation. In conclusion, we have shown that ABA-deficiency in tomato results in priming for pathogen defence responses and that a rapid hyperinduction of ROS-dependent cell wall fortification is a powerful defence strategy to stop the necrotrophic pathogens B. cinerea and E. chrysanthemi.

Author dc:creator

• Asselbergh, Bob

Contributors dc:contributor

• Höfte, M

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

und

Identifier

https://biblio.ugent.be/publication/471805
http://doi.org/1854/8831
https://biblio.ugent.be/publication/471805/file/1880601

OAI identifier oai:identifier

oai:archive.ugent.be:471805