Differential metabolomic responses of PAMP-triggered immunity and effector-triggered immunity in Arabidopsis suspension cells

Introduction

The rhizobacterial tomato pathogen Pseudomonas syringae pv. tomato str. DC3000 (PstDC3000), like many plant pathogenic bacteria, can elicit hypersensitive response in non-host plant cells. PstDC3000 uses a type III protein secretion system (T3SS) to deliver effector proteins.

Objectives

We compared metabolomic responses of Arabidopsis suspension cells to a wild-type PstDC3000, a T3SS deletion mutant PstDC3000D28E, and a pathogen associated molecular pattern (PAMP) flagellin’s N-terminal domain’s 22-aa peptide (flg22) to obtain metabolomics insights into the plant cell PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

Methods

Using targeted HPLC-MRM-MS and untargeted GC-MS approaches, we monitored qualitative and quantitative changes of 312 metabolites in central and specialized metabolic pathways in a time-course study.

Results

The overall metabolomic changes induced by the three treatments included phenylpropanoid, flavonoid, and phytohormone biosynthetic pathways, as well as primary metabolism in amino acid and sugar biosynthesis. In addition to shared metabolites, flg22, PstDC3000D28E and PstDC3000 each caused unique metabolite changes in the course of the development of PTI and ETI.

Conclusion

PstDC3000D28E triggered PTI responses were different from those of flg22. This study has not only revealed the discernible metabolomics features associated with the flg22, PstDC3000D28E and PstDC3000 treatments, but also laid a foundation toward further understanding of metabolic regulation and responses underlying plant PTI and ETI.

     

A genome-scale metabolic model of potato late blight suggests a photosynthesis suppression mechanism

Background

Phytophthora infestans is a plant pathogen that causes an important plant disease known as late blight in potato plants (Solanum tuberosum) and several other solanaceous hosts. This disease is the main factor affecting potato crop production worldwide. In spite of the importance of the disease, the molecular mechanisms underlying the compatibility between the pathogen and its hosts are still unknown.

Results

To explain the metabolic response of late blight, specifically photosynthesis inhibition in infected plants, we reconstructed a genome-scale metabolic network of the S. tuberosum leaf, PstM1. This metabolic network simulates the effect of this disease in the leaf metabolism. PstM1 accounts for 2751 genes, 1113 metabolic functions, 1773 gene-protein-reaction associations and 1938 metabolites involved in 2072 reactions. The optimization of the model for biomass synthesis maximization in three infection time points suggested a suppression of the photosynthetic capacity related to the decrease of metabolic flux in light reactions and carbon fixation reactions. In addition, a variation pattern in the flux of carboxylation to oxygenation reactions catalyzed by RuBisCO was also identified, likely to be associated to a defense response in the compatible interaction between P. infestans and S. tuberosum.

Conclusions

In this work, we introduced simultaneously the first metabolic network of S. tuberosum and the first genome-scale metabolic model of the compatible interaction of a plant with P. infestans.

     

Involvement of the Electrophilic Isothiocyanate Sulforaphane in Arabidopsis Local Defense Responses

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue.Plants are constantly challenged by pathogenic microorganisms and have developed several detection and defense systems to protect themselves against the invaders. Preformed defenses include the waxy cuticle, thick cell walls, and antimicrobial compounds. After recognition of microbe-associated patterns, defense responses are induced, which include the fortification of cell walls and the production of phytoalexins (Monaghan and Zipfel, 2012). Overcoming the preformed and induced defenses of the plant hosts requires adaptation by the pathogen. Pathogenic bacteria use type III secretion to inject proteins (so-called effectors) into the host cytosol in order to overcome plant defense responses (Bent and Mackey, 2007). In turn, plants have developed systems to recognize the pathogenic effectors and mount defense. Recognition of type III effectors by plant resistance (R) proteins induces robust defense responses that frequently include the hypersensitive response (HR).The HR is a complex defense reaction characterized by the induction of programmed cell death (PCD) in the local host tissue as well as the activation of other defense responses in both local and systemic tissue (Mur et al., 2008; Shah, 2009). Oomycetes and true fungi also secrete proteinaceous effectors that can be recognized by host R proteins (Coates and Beynon, 2010; Hückelhoven and Panstruga, 2011; Feng and Zhou, 2012). The lesions formed during the HR vary in size between different host-pathogen pairs; however, a lesion induced at one or a few cells can spread to surrounding cells (Mur et al., 2008). Since pathogens inducing HR typically fail to proliferate, the first infected cell likely releases a compound that promotes PCD in surrounding cells. This is especially clear in models with oomycete and fungal pathogens, where the localization of the pathogen and the spread of cell death around the infection site can be clearly visualized (Mur et al., 2008; Coates and Beynon, 2010). Trailing necrosis is an incomplete resistance phenotype characterized by cell death that trails, but fails to contain, the filamentous growth of the pathogen. One explanation for trailing necrosis is a failure of infected cells to produce a putative mobile defense signal required to enhance defense in neighboring cells. Farther from the site of PCD, other defense pathways are activated and systemic tissue is primed for defense.The hunt for systemically acting compounds has been intense, and several candidates for this signal have been presented (Dempsey and Klessig, 2012). In contrast, even though the phenomenon of HR as a defense reaction was described almost a century ago (Stakman, 1915; Mur et al., 2008), compounds acting on the local tissue scale of the HR have attracted little attention. We set out to find substances released from cells undergoing the HR that could induce cell death in naive tissue. We report that leaf tissue of the model plant Arabidopsis (Arabidopsis thaliana) releases the reactive electrophilic compound sulforaphane after bacterial effector recognition. Mutants affected in sulforaphane production as well as other glucosinolate breakdown products showed delayed or reduced cell death after the recognition of pathogenic effectors and decreased resistance to an oomycete pathogen. Moreover, pretreatment of plants with sulforaphane enhanced resistance against a virulent oomycete isolate. Thus, we interpret this as that sulforaphane and likely similar compounds might both possess direct antimicrobial properties and, through a cytotoxic mechanism, act directly on plant cells to trigger defense responses.