Using DIR1 to investigate long-distance signal movement during Systemic Acquired Resistance

Abstract

During Systemic Acquired Resistance (SAR), a SAR-inducing infection in one leaf initiates movement of phloem-mobile signals to uninfected distant leaves to prime plants to respond in a resistant manner to subsequent infections. Our early work with the dir1-1 (defective in induced resistance) mutant in Arabidopsis demonstrated that the DIR1 protein is required for SAR and led to the hypothesis that DIR1, a lipid transfer protein (LTP), moves to distant leaves to activate SAR. To prove this hypothesis, we monitored DIR1-GFP accumulation in phloem exudates using an estrogen-SAR assay. In this assay, estrogen treatment induces DIR1-GFP expression in one leaf of dir1-1, followed by SAR-induction in the same leaf. DIR1-GFP was detected in exudates collected from local and distant leaves of SAR-induced plants using both DIR1 and GFP antibodies. This provides compelling evidence that DIR1 moves via the phloem to distant leaves to initiate priming. Our work fills a major gap in research on SAR as no other putative SAR mobile signal has been shown to move in planta to distant leaves. To discover how DIR1 enters the phloem, we took advantage of plant lines with compromised cell-to-cell movement caused by overexpression of Plasmodesmata-Located Proteins. These lines were defective for SAR, and DIR1 was not observed in distant leaf phloem exudates, supporting the idea that cell-to-cell movement of DIR1 through plasmodesmata is important for SAR signal movement. To discover new phloem proteins that play a role during SAR, we compared phloem exudate proteomes collected from mock- and SAR-induced leaves using quantitative LC-MS/MS. Numerous proteins were enriched in SAR-induced versus mock-induced phloem exudates and T-DNA knock-out lines in some of these genes were SAR-defective, indicating they contribute to SAR. Identification of SAR-specific phloem proteins may provide clues as to the protein complement of a high molecular weight DIR1-containing complex found in phloem exudates only after SAR induction. We will take advantage of DIR1’s proteinaceous nature to identify proteins in the high molecular weight mobile signal complex, proteins associated with phloem loading of SAR signals and proteins involved in DIR1 perception in distant leaves.

Résumé

Dans le cadre du processus de résistance systémique acquise (RSA), une infection qui induit la RSA dans une feuille amorce l’envoi de signaux qui se propagent par le phloème pour atteindre les feuilles distantes et saines afin d’induire, chez les plants, une réaction de résistance aux infections subséquentes. Nos premiers travaux avec le mutant dir1-1 (déficient en résistance induite) chez Arabidopsis ont montré que la protéine DIR1 était requise pour induire la RSA et ont permis d’échafauder l’hypothèse que la DIR1, une protéine de transfert de lipide (PTL), se propage vers les feuilles distantes pour activer la RSA. Afin de confirmer cette hypothèse, nous avons suivi l’accumulation de DIR1-GFP dans les exsudats de phloème grâce à un biotest œstrogène-RSA. Au cours de ce biotest, le traitement avec œstrogène a induit l’expression de la DIR1-GFP dans une feuille du mutant dir1-1, suivie de l’induction de la RSA dans la même feuille. La DIR1-GFP a été détectée dans les exsudats collectés dans des feuilles voisines et distantes de plants chez lesquels la RSA avait été induite grâce aux anticorps de la DIR1 et de la GFP. Cela prouve irréfutablement que la DIR1 se propage par le phloème pour atteindre les feuilles distantes et stimuler l’amorce de la RSA. Nos travaux comblent une importante lacune quant à la recherche sur la RSA, puisqu’on n’avait jamais démontré qu’un signal putatif de la RSA se propageait in planta vers les feuilles distantes. Pour découvrir comment la DIR1 pénètre dans le phloème, nous nous sommes servis des lignées de plantes dont le mouvement de cellule à cellule est compromis par la surexpression de protéines situées dans les plasmodesmes. Ces lignées étaient déficientes sur le plan de la RSA et nous n’avons pas observé de DIR1 dans les exsudats du phloème des feuilles distantes, ce qui supporte l’idée que la propagation de DIR1 de cellule à cellule par les plasmodesmes est importante quant à la propagation du signal de la RSA. Afin de découvrir de nouvelles protéines du phloème qui jouent un rôle durant la RSA, nous avons comparé, grâce à une analyse quantitative par LC-MS/MS, les protéomes d’exsudats de phloème collectés sur des modèles de feuilles et des feuilles chez lesquelles la RSA avait été induite. De nombreuses protéines ont été enrichies dans les exsudats de phloème découlant de l’induction de la RSA, contrairement à ceux des modèles, et les lignées invalidées affichant des insertions d’ADN-T dans certains de ces gènes n’induisaient pas la RSA. Cela indique que ces protéines contribuent à la RSA. L’identification de protéines phloémiennes propres à la RSA peut fournir des indices quant au complément protéique d’un complexe de poids moléculaire élevé contenant la DIR1, trouvé dans les exsudats de phloème, seulement après induction de la RSA. Nous exploiterons la nature protéique de la DIR1 pour identifier les protéines du complexe de poids moléculaire élevé responsable de la propagation des signaux, protéines associées au chargement des signaux de la RSA dans le phloème ainsi que protéines impliquées dans la perception de la DIR1 dans les feuilles distantes.

Keywords:

• DIR1
• long-distance signalling
• phloem proteins
• plasmodesmata
• Systemic Acquired Resistance

Mots clés:

• DIR1
• plasmodesmes
• proté ines phloémiennes
• résistance systémique acquise
• signal à longue distance

Introduction

The ability of plants to perceive an infection in one tissue and transmit this information to alert or prime distant tissues to respond with broad-spectrum resistance to subsequent infection is known as Systemic Acquired Resistance (SAR). Ever since the first documentation of the SAR phenomenon by Kenneth Chester in 1933 (Chester, 1933), scientists have been interested in understanding how plants communicate over long distances with the hope of using this knowledge to develop disease resistant crops. During the second half of the 20th century, plant pathologists carefully dissected the physiological aspects of SAR in tobacco and cucumber (reviewed in Guedes et al. 1980; Kuc 1982; Tuzun & Kuc 1985). More recently, plant molecular biologists became interested in understanding long-distance signaling mechanisms during SAR. These molecular genetic studies were facilitated by the development of the Arabidopsis–Pseudomonas syringae SAR model (Cameron et al. 1994). A key feature of SAR is the long-distance movement of signals from induced to distant leaves via the phloem. In distant leaves, signals are perceived and plants are primed to respond to virulent infections in a resistant manner (reviewed in Champigny & Cameron 2009). The primed state is associated with the accumulation of inactive protein kinases and chromatin modification to SAR-associated gene promoters, all of which are thought to provide a molecular memory of the initial infection (reviewed in Conrath 2011). Upon subsequent infection, primed distant leaves respond rapidly and effectively by accumulating the defense compound salicylic acid (SA) to initiate the interaction of NPR1 and TGA transcription factors to up-regulate SAR defence gene expression (Yan & Dong 2014).

DIR1 moves to distant tissues during SAR

Our early work indicated that the lipid transfer protein (LTP) DIR1 is required for the production or transport of SAR long-distance signals in Arabidopsis (Maldonado et al. 2002). Lascombe et al. (2008) resolved the crystal structure of DIR1 and demonstrated that like most LTPs, DIR1’s four disulphide bonds allow it to fold to produce an internal hydrophobic cavity that binds two lipids in vitro. At this time, we also demonstrated that DIR1 is detected in phloem sap-enriched petiole exudates of SAR-induced, but not mock-inoculated leaves. This led us to hypothesize that DIR1 is activated upon SAR induction, allowing it to bind and chaperone a lipid ligand to distant leaves to initiate priming. Alternatively, a DIR1-lipid complex may act as a long-distance SAR signal. Proving that a protein or molecule is a mobile signal is challenging for two main reasons. First, the exogenous application of mobile signal candidates in leaf infiltration experiments is technically difficult, as care must be taken to limit infiltration of signals into a single leaf. A bigger challenge is that some molecules enter the phloem after infiltration into the leaf intercellular space, leading to false positive results (Moore et al. 2006; Rocher et al. 2006; Zeidler et al. 2010). Therefore, this method alone is insufficient to definitively demonstrate that a candidate signal moves to distant tissues. Second, the capacity for signal production must be limited to the SAR-induced leaf to definitively demonstrate that the presence of a candidate signal in distant leaves is due to long-distance movement rather than de novo biosynthesis caused by the perception of actual SAR signals. Ideally, this would be achieved by grafting or using a transgenic/mutant line that is incapable of producing the candidate signal until prompted to do so by using an inducible promoter system (Champigny et al. 2013). A number of small molecules (methyl SA, glycerol-3-phosphate derivative, dehydroabietinal, azelaic acid, pipecolic acid) have been identified as SAR long-distance signal candidates in Arabidopsis and tobacco (reviewed in Dempsey & Klessig 2012; Shah et al. 2014); however, only DIR1 has been shown to move to distant leaves during SAR in Arabidopsis (Champigny et al. 2013). To demonstrate this, we developed the estrogen-SAR assay to restrict DIR1-GFP expression to one leaf by creating a transgenic line that expresses DIR-GFP under the control of the XVE estrogen-inducible promoter (Zuo et al. 2000) in the dir1-1 mutant background. First, we demonstrated that DIR1-GFP expression was limited to just the estrogen-treated leaf. Second, DIR1-GFP expression was induced by estrogen treatment, followed by mock- or SAR-inducing the same leaves. Third, long-distance DIR1-GFP movement was monitored by collecting phloem exudates from induced and distant leaves followed by immunoblot analyses using DIR1 and GFP antibodies. DIR1-GFP was not detected in exudates collected from mock-induced plants, but was detected in phloem exudates from induced and distant leaves of SAR-induced plants. Our study provides compelling evidence that Arabidopsis DIR1 (AtDIR1) is activated to move from induced to distant leaves during SAR and is therefore a SAR mobile signal or signal chaperone (Champigny et al. 2013). Two tobacco DIR1 orthologues have also been shown to be important for the SAR response (Liu et al. 2011) and a DIR1 protein has been observed in tomato phloem exudates (Mitton et al. 2009). We identified DIR1 orthologues in tomato, cucumber and soybean and demonstrated that Agrobacterium-mediated transient expression of two cucumber DIR1 othologues complements the Arabidopsis dir1-1 SAR defect. Moreover, we observed that cucumber phloem exudates contain a DIR1-type protein that rescued the SAR defect of dir1-1 in experiments using our cucumber-Arabidopsis model system (Isaacs et al. Forthcoming). These experiments provide compelling evidence that the DIR1-mediated SAR response is conserved in economically important crops.

Using DIR1 as a tool to elucidate SAR

The resistance-promoting activity of azelaic acid, a glycerol-3-phosphate derivative, and dehydroabietinal require functional AtDIR1 (Jung et al. 2009; Chanda et al. 2011; Chaturvedi et al. 2012). Additionally, the SAR-related LTPs, Azelaic acid Induced1 (AZI1) and Early Arabidopsis Aluminium Induced1 (EARLI1) interact with AtDIR1 in transient expression experiments in Nicotiana benthamiana (Yu et al. 2013; Cecchini et al. 2015). These findings suggest that AtDIR1 participates as a member of a SAR signal complex. The recent discovery of a AtDIR1-containing high molecular weight protein complex in phloem exudates collected from SAR-induced leaves (Chaturvedi et al. 2012; Shah et al. 2014) supports this idea and indicates that AtDIR1 plays a central role during SAR. We will take advantage of AtDIR1’s proteinaceous nature and role in both SAR-induced and distant leaves by using protein interaction assays/screens to understand and link together both ends of the SAR pathway. For example, what activates AtDIR1 for movement out of SAR-induced leaves? We hypothesize that AtDIR1 interacts with proteins that restrict its movement until a leaf has been induced for SAR and/or interacts with proteins that promote its movement. Moreover, we can also use AtDIR1 as bait to identify interacting proteins in distant leaves to understand how the AtDIR1-containing SAR signal complex transduces news of the initial infection to distant leaf cells to induce the primed state. Finally, total protein levels are higher in phloem exudates collected from SAR-induced versus mock-inoculated leaves (Champigny et al. 2013; Carella et al. 2015a), suggesting that a number of phloem-localized proteins are required for the formation or maintenance of a SAR signal complex during long-distance movement to distant leaves. The accumulated evidence supports the idea that AtDIR1 is a member of this complex, making AtDIR1 an excellent probe to identify phloem-localized proteins involved in SAR.

Investigating the role of plasmodesmata in SAR mobile signal movement

Little is known about the physiological mechanisms and routes of signal movement during SAR. Studies in tobacco, cucumber and Arabidopsis indicate that SAR mobile signals move predominately via the phloem, but also cell-to-cell (reviewed in Champigny & Cameron 2009). In plants, the cell-to-cell (symplastic) movement of macromolecules occurs via membrane-lined plasmodesmatal junctions that cytosolically connect neighbouring plant cells (Burch-Smith & Zambryski 2012). These junctions provide a conduit for cell-to-cell movement between companion cells and sieve elements, implicating plasmodesmata as important regulators of long-distance movement of macromolecules through the phloem. We hypothesized that SAR mobile signals move through plasmodesmata to access the phloem for long-distance movement during SAR.

To investigate if movement through plasmodesmata is important for SAR mobile signaling, we examined the impact of restricting plasmodesmatal pore size on long-distance movement of AtDIR1 during SAR in Arabidopsis. This was achieved using recently created transgenic lines that overexpress members of the Plasmodesmata-Located Protein (PDLP) family, PDLP1 and PDLP5. These PDLP proteins have been shown to accumulate at plasmodesmata and, when overexpressed, reduce the cell-to-cell movement of fluorescent dyes or proteins (Thomas et al. 2008; Lee et al. 2011). Cell-to-cell movement through plasmodesmata is important for the loading and unloading of macromolecules into and out of the phloem. Therefore, we hypothesized that PDLP1/5 overexpression would impair the long-distance movement of AtDIR1, leading to a defect in SAR. As hypothesized, both PDLP1- and PDLP5-overexpressing lines were defective in the manifestation of SAR (Carella et al. 2015a). In wildtype Col-0, AtDIR1 antibody signals were observed in phloem exudates collected from local and distant leaves of SAR-induced but not mock-inoculated plants. In contrast, AtDIR1 antibody signals were not detected in phloem exudates collected from distant leaves of SAR-induced PDLP1/5-overexpressing plants (Carella et al. 2015a). The results demonstrate that the overexpression of PDLP1 or PDLP5 suppresses the long-distance movement of AtDIR1 during SAR and suggest that regulation of plasmodesmatal pore size is important for long-distance SAR signalling.

Identification of phloem proteins important for SAR

During SAR, total protein levels rise in phloem exudates (Champigny et al. 2013; Carella et al. 2015a), suggesting that numerous proteins may be important for the formation or maintenance of a SAR mobile signal complex that moves from induced to distant leaves. We used quantitative label-free LC-MS/MS to perform comparative proteomics in collaboration with Drs Vlot and Merl-Pham of the Helmholtz Zentrum in Germany. We compared phloem proteomes obtained from Arabidopsis plants that were mock inoculated or induced for SAR with virulent and avirulent P. syringae pv. tomato strains to identify proteins specific to SAR. We are currently validating our SAR phloem proteome by determining which of these proteins contribute to SAR. To do this, T-DNA mutants in phloem protein genes were obtained and assayed for SAR competence. We identified a number of proteins that are required for the manifestation of SAR, including thioredoxins. Given that AtDIR1 has four disulphide bonds, it is possible that thiol and disulphide conversion by thioredoxins is required for AtDIR1 function in the phloem. As discussed above, evidence from a number of labs suggests that AtDIR1 is part of a large protein complex in the phloem; therefore, we will use AtDIR1 to determine if any of the SAR-related phloem proteins are members of the AtDIR1-containing SAR mobile complex.

Current SAR model

Evidence from a number of labs supports the idea that DIR1 is a central player in SAR and that DIR1 accesses the phloem for long-distance movement from induced to distant leaves. Our work with PDLP-overexpressing lines indicates that cell-to-cell movement through plasmodesmata is important for movement of DIR1 to distant leaves; however, the precise cellular/molecular mechanisms associated with this movement remain unexplored. We propose a model for SAR mobile signal movement (see diagram in Carella et al. 2015b) in which mesophyll cells produce intracellular and/or extracellular signals in response to SAR-inducing pathogens. These signals move symplastically (via plasmodesmata) or apoplastically to access the vasculature (Carella et al. 2015b). Given the number of putative small molecule SAR signals (Dempsey & Klessig 2012), it is plausible that some SAR signals access the phloem via the symplastic route, while others use the apoplastic route. However, since proteins and larger macromolecules such as RNA and viral genome complexes are believed to access the phloem symplastically from companion cells (Turgeon & Wolf 2009; Hipper et al. 2013), we hypothesize that larger SAR signals or complexes access the phloem symplastically. Upon entering the phloem, small mobile SAR signals may move independently or as part of the DIR1-containing protein complex from locally infected to distant leaves. We have laid the groundwork for understanding how SAR signal complexes are formed and maintained during movement in the phloem by analysing the SAR phloem proteome. It is well established that SAR signals/complexes arrive in distant leaves; however, we know little about the dissemination of SAR mobile signals/complexes within distant leaves. We hypothesize that upon arrival in distant leaves, mobile SAR signals and/or complexes are symplastically unloaded from sieve elements to companion cells, and then from companion cells to phloem parenchyma, similar to other phloem mobile macromolecules (Imlau et al. 1999; Baluska et al. 2001). Once inside phloem parenchyma, long-distance SAR signals may move to other leaf cell types to induce SAR. Alternatively, the unloading of SAR signals in distant leaves may lead to the generation of secondary signals that communicate with distant mesophyll cells, either through plasmodesmatal cell-to-cell movement or via the apoplast.

Applying knowledge of DIR1 and SAR to improve disease resistance

Over the last several years, the induced resistance research area has made significant progress and although many questions remain to be addressed, the field has matured, allowing applied and basic researchers to collaborate to design and implement practical solutions to modern agriculture issues. Identifying economically effective and environmentally friendly methods of pest control for field and greenhouse crops is increasingly important to producers and consumers alike. Researchers have discovered a number of small molecules that tap into the SAR pathway to initiate disease resistance without compromising normal growth and development. These include the resistance-inducing putative SAR mobile signals azelaic acid, glycerol-3-phosphate, pipecolic acid, dehyroabietinal and methylsalicylic acid (Shah et al. 2014). Moreover, small molecules like β-aminobutyric acid (BABA), folic acid and vitamin B1/2, act at other stages to induce SAR-like resistance (reviewed in Beckers & Conrath 2007).

In the past, synthetic SA-analogues have been used to induce resistance in crops; however, this resistance was often associated with defects in plant growth and the suppression of JA-mediated resistance to herbivores. In contrast, a recent study demonstrated that non-toxic, edible and biodegradable glycerol sprays stimulated short-term plant growth and disease resistance. During this process, glycerol was converted to glycerol-3-phosphate, stimulating SAR-associated gene expression and disease resistance (Zhang et al. 2015). We have developed a cucumber-Pseudomonas SAR model to identify small molecules that protect cucumber plants from pathogen infection without detrimental effects on growth or herbivore resistance. In collaboration with plant pathologists, the ability of SAR-inducing molecules discovered in the lab will be tested to identify chemicals that provide effective and environmentally sustainable resistance in the greenhouse and field.

We will also combine our basic knowledge of DIR1’s central role in long-distance SAR signalling to identify novel immune-priming small molecules using a chemical biology approach. To do this, we are developing a high-throughput chemical screen to identify environmentally friendly chemicals that activate DIR1 for movement to distant leaves to establish SAR.

Concluding remarks

This is an exciting time for basic and applied research on induced disease resistance. Our knowledge of basic plant defence mechanisms and continued advancements in applying this research has reached a critical stage. The feasibility of applying our basic knowledge to improve disease resistance is becoming a reality. Moreover, it is becoming increasingly important to understand how crop plants deal with concurrent and variable abiotic and biotic stresses throughout the growing season. Basic and applied research focused on understanding how multiple stresses affect greenhouse and field crop plants is an additional area that will contribute to improving disease resistance. As demonstrated through this symposium, opportunities for collaboration between applied and basic induced resistance researchers are on the rise and will greatly aid in identifying sustainable methods to control disease and increase crop yields.

Acknowledgements

We would like to thank Dr Deena Errampalli for bringing together the speakers for this symposium and the Canadian Phytopathological Society and Canadian Society of Plant Biology for the sponsoring this symposium as part of Botany 2015.

Additional information

Funding

This work was funded by NSERC Discovery, RTI and CFI Leadership grants to RKC and Deutsche Forschungsgemeinschaft (DFG) grant to ACV, and an Ontario Graduate Scholarship to PC.