Julie GERVAIS & Yohann PETIT attended the IS-MPMI XVIII Congress Portland, USA

Following Dr Marie-Hélène Balesdent's request :
two students Julie GERVAIS (INRA BIOGER, France) and Yohann PETIT (INRA BIOGER, France) attended the  IS-MPMI XVIII Congress (Portland, USA – 17-21 July 2016).


They were partially sponsored by GCIRC to attend the event.

Here is their report : 

My name is Yohann Petit, and I am currently in my third year of thesis in the French National Institute of Agronomical Research. I am working on the functional characterization of some fungal virulence factors. During plant infection, pathogens secrete an arsenal of effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Fungal effector genes typically encode small proteins, predicted to be secreted (SSPs, Small Secreted Proteins), with no homology in databases, and absence of known motif. As such their function or role in pathogenesis is mostly unknown. The phytopathogenic ascomycete Leptosphaeria maculans is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 10 of them having been demonstrated as being effectors. The objective of my PhD project is to elucidate the involvement of L. maculans effectors into pathogenicity through their structural and functional characterization and identification of their interactants. This characterization includes determination of subcellular localisation of those effectors, search for plant targets and identification of cellular processes targeted.

I am Julie Gervais and am a third year PhD student in INRA BIOGER. Thank to the GCIRC travel award, I was able to give a talk at the IS-MPMI XVII Congress on my thesis work which is focused on the fungal pathogen of oilseed rape, Leptosphaeria maculans, responsible for the stem canker disease. This fungus has two colonization stages of the plant.  During the first stage, the fungus infects leaves and cotyledons. Once in the leaves, the fungus has a short biotrophic stage of 10 days and then switches to necrotrophy. Following this primary leaf infection, L. maculans grows inside the stem tissues during a long endophytic systemic colonization. This colonization is completely symptomless and may last up to 9 months. I am aiming to gain a better understanding of how the fungus can grow inside the oilseed rape stem for several months without causing any symptom. I am particularly focused on the identification of new effectors, secreted proteins, produced by the fungus enabling it to develop itself efficiently into the plant.

The GCIRC travel award allowed us to participate to the 17th congress of International Society for Molecular Plant-microbe Interactions (IS-MPMI) in Portland (17-21 July 2016) and present our thesis work. This conference was also an opportunity to discuss with international scientists and learn about current research in the plant-microbe interaction field. The IS-MPMI congress regroups every two years more than 900 scientific in the field of plant-microbe interactions. A lot of thematics are addressed like genomic of plant pathogens, functional analyses of plant-microbe interactions, breeding for resistant plant and plant biotechnology.

We are presenting here a report of the Congress MPMI divided in three parts. The first part is a focus on the pathosystem Leptosphaeria maculans/Brassica napus. Then we report the presentations of two concurrent sessions we were especially interested in: impacts of vesicular trafficking and cytoskeleton dynamics on microbe-host interactions (Concurrent session 11) and molecular plant-microbe dialogue in the apoplastic space during infection (Concurrent session 20).

The pathosystem Leptosphaeria maculans / Brassica napus

The Canadian team of D. Fernando presented a poster on the identification of a new subclade of Leptospaheria biglobosa isolated on B. rapa and named L. biglobosa americensis. By PCR approaches on the genes ITS, actin and β-tubuline, they established phylogenic relations between L. biglobosa canadensis and L. maculans brassicae. This team demonstrated that L. biglobosa americensis was more virulent on the cultivar Westar than other L. biglobosa. L. biglobosa americensis led to more important lesions on Brassica rapa, oleracea and juncea.

D. Fernando team also presented a poster on hormonal regulations in B. napus in interaction with L. maculans. They showed that genes potentially involved in biosynthetic and signaling pathways of salicylic acid and ethylene were especially over-expressed during the biotrophic colonization of oilseed rape. They also showed that the quantitative resistance, compared to the qualitative resistance was more correlated with the over-expression of genes involved in the jasmonic acid signaling pathways and MAPKKK pathways.

N. Larkan and M. Borhan reported the identification of QTL conferring resistance to L. maculans in spring cultivars. These results were published just after the congress in the revue BMC Plant Biology. This team also worked on transcriptomic approaches during the colonization of cotyledons, petioles and stems of oilseed rape in two spring cultivars (with and without quantitative resistance) to better understand the interaction between the pathogen and the plant.

With these different presentations, this congress allowed us to learn about current work on the pathosystem L. maculans / B. napus.

 Impacts of vesicular trafficking and cytoskeleton dynamics on microbe-host interactions (Concurrent session 11)

Continuously threatened by infectious pathogens from the environment, plants have evolved strategies to defend themselves.  This session summarized recent studies dealing with vesicular trafficking and cytoskeleton dynamics during host-microbe interactions.

Early immune responses are initiated by direct contact of microbes or microbe-derived patterns with  pattern recognition receptors (PPRs) at the plasma membrane of plant cells (Smith et al., 2014). Little is known about the components of vesicular trafficking that contribute to PPR trafficking to and from the plasma membrane. The Group of Dr. Antje Heese (University of Missouri, USA) focuses their work on clathrin-coated vesicles (CCVs), which are involved in intracellular cargo transport at the plasma membrane and the trans-Golgi network (TGN), and studies the role of CCVs in plant immunity. They showed evidence of epsin N-terminal homolog (ENTH) domain proteins contributing to CCV formation. In their study, the enth-2 mutant Arabidopsis showed reduced MAMP-related responses and a decreased level of the PPR FLS2 protein abundance at the plasma membrane.

A main goal of Dr. Christopher Staiger’s group (Purdue University, USA) was to determine if cytoskeletal rearrangement is involved in PAMP-triggered immunity (PTI) in Arabidopsis (Li et al., 2015). They showed that the movement of actin filaments can be divided into three phases including convolutedness, elongation and severing. In addition, their findings demonstrated that MAMP-related responses like Ca2+ transients and accumulation of reactive oxygen species (ROS) led to increased density of actin filaments. Actin assembly is regulated by several innate immune responses that target capping proteins (CPs). One of the key findings highlighted the necessity for the interaction of phosphatidic acid (PA) and CP for the immediate cytoskeletal responses that take place during the immune response.

Immune responses like the recognition of PAMPs at the plasma membrane are taking advantage of the microtubule network to propagate defense signals to diverse cellular compartments. The microtubule network is also used to secrete defense compounds like antimicrobial compounds or other defense-related molecules. HopE1 is a Pseudomonas syringae type III effector with PTI-suppressing activity that significantly contributes to virulence. Dr. Ming Guo (University of Nebraska, USA) from the Alfano laboratory reported that HopE1 interacts with the plant protein MAP65-1, which is implicated in microtubule crosslinking in a calmodulin-dependent manner. Analyses of Arabidopsis lines either overexpressing HopE1 or deleted for MAP65-1 showed that HopE1 dissociates MAP65-1 from the microtubule network, leading to microtubule network disruption. HopE1 has also been shown to inhibit PR-1 secretion. Together those results support a model where HopE1 interferes with PTI-associated trafficking by disrupting the microtubule network leading to interruption of immune signaling and cell-wall associated defenses (Guo et al., 2016).

In barley, the protein RACB is required for susceptibility to the fungal pathogen Blumeria graminis f. sp. hordei (Bgh) (Opalski et al., 2005). The team of Dr. Christopher McCollum (Technical University of Munich, Germany) showed that the Bgh effector ROPIP1 interacts with RACB and induces its activation. They demonstrated that the activated RACB impacts the cellular localization of three ROP inducing proteins (RIPs), leading to microtubule depolymerization, probably through the kinase 13A (Mucha et al., 2010). They also showed that overexpression of one of the RIPs leads to increased susceptibility of barley to Bgh. This work suggests that Bgh may manipulate RACB to interfere with microtubule organization.

All in all, the presenters from this session were able to give deep insights regarding the impact of vesicular trafficking and cytoskeletal dynamics on plant immunity and the significance of endocytic and exocytic processes in evolving resistance to pathogens.

 References

Guo, M., Kim, P., Li, G., Elowsky, C.G., and Alfano, J.R. 2016. A Bacterial Effector Co-opts Calmodulin to Target the Plant Microtubule Network. Cell Host Microbe. 19:67–78.

Li, J., Henty-Ridilla, J.L., Staiger, B.H., Day, B., and Staiger, C.J. 2015. Capping protein integrates multiple MAMP signalling pathways to modulate actin dynamics during plant innate immunity. Nat. Commun. 6:7206.

Mucha, E., Hoefle, C., Hückelhoven, R., and Berken, A. 2010. RIP3 and AtKinesin-13A - a novel interaction linking Rho proteins of plants to microtubules. Eur. J. Cell Biol. 89:906–916.

Opalski, K.S., Schultheiss, H., Kogel, K.-H., and Hückelhoven, R. 2005. The receptor-like MLO protein and the RAC/ROP family G-protein RACB modulate actin reorganization in barley attacked by the biotrophic powdery mildew fungus Blumeria graminis f.sp. hordei. Plant J. Cell Mol. Biol. 41:291–303.

Smith, J.M., Leslie, M.E., Robinson, S.J., Korasick, D.A., Zhang, T., Backues, S.K., Cornish, P.V., Koo, A.J., Bednarek, S.Y., and Heese, A. 2014. Loss of Arabidopsis thaliana Dynamin-Related Protein 2B reveals separation of innate immune signaling pathways. PLoS Pathog. 10:e1004578.

 Molecular plant-microbe dialogue in the apoplastic space during infection (Concurrent session 20)

The apoplastic space serves as one of the first interaction sites between plants and microbes (Doehlemann and Hemetsberger, 2013). Those interactions include recognition of microbe-associated molecular patterns (MAMPs) by pattern recognition receptors (PRRs) and the subsequent immune responses like secretion of antimicrobial compounds. Pathogens in turn secrete effector proteins in order to circumvent plant defenses (Macho and Zipfel, 2014).

Dr. Thorsten Nürnberger (University Tübingen, Germany) reported on two novel leucine-rich receptor proteins RLP23 and RLP32 that are able to sense protein PAMPs from various microbes and contribute to plant innate immunity (Albert et al., 2015). Both PRRs were shown to constitutively interact with and require SOBIR1 as a co-receptor. Another highlight of the group’s findings was that the co-receptor BAK1 seems to be recruited into the RLP/SOBIR1 complex in a ligand-dependent manner. Additionally, the two RLPs are specific to Brassicaceae. In their studies, they showed evidence that RLP23 recognizes a peptide motif within the necrosis-and-ethylene-inducing protein 1 like proteins (NLPs). On the other hand, RLP32 recognizes protein patterns from the bacteria Ralstonia solanacearum or Escherichia coli.

The apoplastic pathogenesis-related protein 1 (PR-1) was discovered in 1970 in pathogen-challenged tobacco plants (Van Loon and Van Kammen, 1970). An antimicrobial activity was proposed for this protein and the PR-1 gene is often used as a marker for defense-related responses. Despite its importance in defense, no mode of action has been found for PR-1. PR-1 is a member of the CAP-family of proteins involved in sterol-binding, inciting Dr. Jordi Gamir (University of Fribourg, Germany) to hypothesize a sterol-binding activity for PR-1. This hypothesis was validated by the finding that PR-1 complements yeast pry1 and pry2 (pathogen-related yeast) mutants, allowing restoration of sterol export. Moreover, an in vitro interaction was demonstrated between PR-1 and diverse sterols. This sterol-binding activity is necessary for PR-1 antimicrobial activity, as shown by the titration of its antimicrobial activity by addition of sterols. Two modes of actions of PR-1 can thus be hypothesized: either PR-1 interferes with sterol uptake by the pathogen or it directly binds sterols of the pathogen’s plasma-membrane to cause damage.

Nematodes are known to infect both plants and animals, but the way they suppress the immune response is poorly understood. The group of Dr. Lozano-Torres (Wageningen University, The Netherlands) recently showed that the apoplastic effectors, venom allergen-like proteins (VAPs), are able to repress plant defense response (Lozano-Torres et al., 2014). By expressing heterologous VAPs from nematodes in plants, they observed increased susceptibility toward various pathogens, indicating a compromised plant basal immunity. The induction of ectopic VAPs seems to act on cell surface localized proteins. Furthermore, their release into plants tissue is synchronized with the enzymatic breakdown of the plant cell wall. Taken together, these results suggest that VAPs inhibit early activation of immune responses and damage plant tissue.

Dr. Lay-Sun Ma (Max Planck Institute for Terrestrial Microbiology, Germany) from Dr. Regina Kahmann’s laboratory reported the identification of a new apoplastic effector Rsp3 from the fungal pathogen Ustilago maydis. Rsp3 was specifically expressed during infection and required for U. maydis virulence. Ma demonstrated that Rsp3 attached to the outside of fungal hyphae and interacted with maize DUF26 domain-containing proteins that belong to a family of antifungal proteins. The antifungal activity of the DUF26 proteins was blocked by Rsp3. Results thus suggested that the apoplastic effector Rsp3 protects U. maydis from host antifungal activity, presumably by inhibiting DUF26 domain-family proteins from binding to the fungal cell wall.

To summarize, speakers of this session presented evidence for a large array of strategies associated with the plant-pathogen interactions in the apoplastic space, either from the plant perspective with RLPs and secretion of PR proteins or from the pathogen side with various apoplastic effector proteins.

 

References

Albert, I., Böhm, H., Albert, M., Feiler, C.E., Imkampe, J., Wallmeroth, N., Brancato, C., Raaymakers, T.M., Oome, S., Zhang, H., et al. 2015. An RLP23-SOBIR1-BAK1 complex mediates NLP-triggered immunity. Nat. Plants. 1:15140.

Doehlemann, G., and Hemetsberger, C. 2013. Apoplastic immunity and its suppression by filamentous plant pathogens. New Phytol. 198:1001–1016.

Lozano-Torres, J.L., Wilbers, R.H.P., Warmerdam, S., Finkers-Tomczak, A., Diaz-Granados, A., van Schaik, C.C., Helder, J., Bakker, J., Goverse, A., Schots, A., et al. 2014. Apoplastic venom allergen-like proteins of cyst nematodes modulate the activation of basal plant innate immunity by cell surface receptors. PLoS Pathog. 10:e1004569.

Macho, A.P., and Zipfel, C. 2014. Plant PRRs and the Activation of Innate Immune Signaling. Mol. Cell 54:263–272.

Van Loon, L.C., and Van Kammen, A. 1970. Polyacrylamide disc electrophoresis of the soluble leaf proteins from Nicotiana tabacum var. “Samsun” and “Samsun NN.” Virology. 40,:199–211.