Final Report Summary - SIGNSTRESSPATH (Networking by stress signalling pathways: identification of novel regulators of combinatorial stress tolerance)
Plant species are continuously attacked by harmful microbial pathogens. Besides biotic stressors, plants have to deal with extreme conditions such as drought, heat, cold, water logging, high salinity or toxic compounds. All these stress situations must trigger signals that alter the plant’s physiology and growth to ensure survival in hostile environments. The plant hormones salicylic acid (SA), jasmonic acid (JA), ethylene (ET), and abscisic acid (ABA) emerged as the main players in coordinating signalling networks involved in the adaptive response of plants to its (a)biotic environment. Changes in hormone concentration or sensitivity, which can be triggered under (a)biotic stress conditions, mediate a whole range of adaptive plant responses. In presence of simultaneous stresses, the signalling cascades that are regulated by these hormones partly overlap and are interconnected in a complex network of cross-communicating pathways. The great regulatory potential of such a network allows plants to quickly adapt to their (a)biotic environment and to utilize their resources in a cost/energy-efficient manner. While the response of plants to single stress conditions has been relatively well studied, our understanding of complex multi-dimensional signal communication during the interaction of plants with multiple stresses is still at its infancy.
Summary of Project objectives: The research focussed on the molecular responses triggered by drought and the necrotrophic pathogen Botrytis cinerea, two among the most severe stresses that every year cause significant economic losses both in the field and in postharvest conditions. The project aims to gain an integrated understanding of the interactions between biotic and abiotic stress molecular pathways and identify novel properties of the signalling circuitry that regulates the adaptive response to multi-stress situations. The whole project is organized along two main objectives (O1,O2), plus an additional one (AO):
O1- Multi-stress gene regulatory networks: discovering novel signalling nodes involved in the response of plants to combinatorial stresses. By a combination of state-of-the-art multidisciplinary approaches the molecular responses triggered by the single biotic/abiotic stress or by their combination have been investigated in the plant model Arabidopsis thaliana.
O2- Functional characterization of master switches of cross-talk pathways. The predicted key genes of the cross-talk between biotic/abiotic stress were deeply characterized.
AO- Identification of novel regulators of combinatorial hormonal stress response. Another way with which mechanisms for combinatorial stress resistance can be unravelled, is through looking at specific defense hormone signaling pathways. So, an additional work package was included in the project with the aim to identify key regulators of the cross-talk between phytohormones mimicking combinatorial stress under study.
Description of the work performed since the beginning of the project:
O1- The whole-transcriptome profile of Arabidopsis triggered by the chosen single and combined abiotic and biotic stresses was analyzed by RNA sequencing (RNA-Seq). To study the effect of drought stress on disease resistance to the necrotrophic fungus, a set of Arabidopsis ecotype Col-0 plants were treated with the pathogen B. cinerea, drought, a combination of the two stresses and not treated (control), and leaves were harvested at different time points. Then the whole genome transcript profiling was performed by using high throughput new generation sequencing platforms (NGS). Bioinformatic analysis allowed to translate the output of the sequencing in quantification of transcript abundance. Statistically significant differentially expressed genes (DEGs) in single stress/control and double stress/control were taken into account for the further analysis. Then, the mathematical modelling of DEGs, in combination with Gene Ontology analysis were used to highlight biological processes and molecular mechanisms differently altered by single and double stresses.
O2- Clusters of DEGs identified by the modelling as potentially having an important role in the interaction between the single and double stress, were further investigated. In particular, among them genes predicted as key hubs of the cross-talk between the two stresses were functionally characterized under a system biology perspective and their role in the multi-stress condition was studied in depth.
AO- Exploiting natural variation of Arabidopsis thaliana in a Genome wide association (GWA) mapping study is a powerful approach to unravel novel genes involved in the hormonal cross-talk regulating the multiple stress responses. In the work carried out, the source of natural variation is represented by a collection of 349 Arabidopsis thaliana accessions collected worldwide (Hapmap collection, genotyped for 250000 SNPs), that were screened for combinatorial hormonal treatment by analysing the expression of a selected marker gene. In particular, JA+SA and ABA+JA, both involved in the cross-talk between drought and B. cinerea were chosen. Gene expression data were then used as input in algorithms developed for GWA analysis, able to calculate the statistical association between the input and SNPs significantly correlated with the process under study. Based on the location of the SNPs, putative key genes involved in hormonal cross-talk were selected and their role in the phenomenon under study experimentally validated.
Description of main results achieved:
O1-O2: Collectively, our transcriptome data provided a powerful resource to study plant responses during multifactorial stress, highlighting biological processes and molecular mechanisms involved in the interactions of plants with their environment.
Comparative analysis of plant gene expression triggered by single and double stress revealed a complex transcriptional reprogramming and rewiring. Mathematical modelling of transcriptomic data, in combination with Gene Ontology analysis identified primary and secondary metabolic as well as defence related processes specifically affected by single and double stresses. Briefly, B. cinerea strongly induced clusters of genes specifically involved in ethylene and chitin response, as well as SA pathway and the camalexin biosynthesis, among the most prominent responses usually activated against necrotrophs. On the other hand, the drought treatment strongly repressed these responses. This explains the results of the performed phenotypic analysis that clearly showed an increasing susceptibility to B. cinerea in plants subjected to drought. Then, the repression of SA pathway by drought looked so interesting that we decided to further investigate it. A down-regulation of PR-1 and WRKY70 genes caused by drought suggested that NPR1, a key nodal regulator of SA pathway might play a central role in drought-B. cinerea cross-talk. The role of NPR1 in hormonal cross-talk regulating the stress under investigation have been studied, demonstrating that NPR1 is a major player in SA signaling as well as in suppression of JA responses by SA. . Its role in drought-B. cinerea is currently under investigation.
AO: From the GWA study, 7 genes have been predicted as candidates for SA/JA cross-talk and 17 for ABA/JA cross-talk. They play a role in a broad range of different biological processes that go from signal transduction to macromolecules biosynthesis. One of the most interesting candidate genes for SA/JA is glyI4, a gene involved in the metabolism of methylglyoxal, a compound produced in defense responses. This gene is known to be involved in abiotic stress responses as well as cytokinin and ABA signaling pathway. For ABA/JA Atsam2, a methionine adenosyltransferase and oct6, a carbohydrate membrane transporter were among the most promising. Both are involved in abiotic stress responses and moreover, among the predicted interactors of oct6 there is JAZ10, a negative regulator of JA signaling pathway. The corresponding T-DNA lines have confirmed the role as positive regulator of SA/JA cross-talk for glyI4 and negative regulator of ABA/JA cross-talk for both Atsam2 and oct6. Moreover, looking at the RNA-Seq data all genes have been found to be affected by combined treatment, compared to the single one, strengthen the relevance that the hormonal signaling has on regulating multiple biotic/abiotic stress responses.
Conclusions and potential impact and use of the results:
So far, only a few studies have addressed plant responses to different stress factors occurring simultaneously and analyzed combined stress situations. Even less is known about how a combined stress can affect the plant transcriptome profile different than the single stresses alone. In this perspective, it is expected that results will increase our understanding of the natural mechanisms used by plants to resist to multiple adverse conditions and may open up new possibilities for rewire defence networks through modulating hormone-regulated signalling pathways, minimizing impact of stresses on crop yield.
Moreover, the results that come out from the project will not only consolidate our understanding of mechanisms underlying biotic and abiotic stress resistance and will produce a wealth of scientific information, but it will also offer transferable skills to the plant breeding industry or agricultural biotech companies. For instance, regulatory genes of multiple stress responses could be used in marker-assisted breeding programmes, or could be exploited in cis-genic approaches to engineer second-generation GM crops that require less or none chemical agents to control diseases.