Research Group El Kasmi
NLR Biology and the relationship of Immunity and Trafficking
Dr. Farid El Kasmi
Junior Research Group Leader
University of Tübingen
Auf der Morgenstelle 32
Phone: +49 7071 29 7 88 82 (office)
+49(0)7071 29-73290 (lab)
Fax: +49 (0)7071 - 29 32 87
Plants have evolved sophisticated mechanisms to fight off pathogenic microbes or to establish mutualistic relationships with beneficial ones. In both interactions a plant cell has to initiate an appropriate signaling cascade and to reprogram cellular processes in a way the whole plant can either gain most out of the encounter or prevent a hostile invasion. Microbial organisms use ‘effector’ proteins to manipulate and modify a plant cells response in favor of their survival. It still remains largely elusive how effector proteins perturb specific cellular processes, such as intracellular protein and membrane trafficking, and how plants respond to such an effector-induced manipulation. Plants possess intracellular immune receptors, so called NLR proteins, that may directly or indirectly recognize effectors and upon activation induce a defense response. Some NLRs are membrane localized and it is still unknown how they initiate a transcriptional reprogramming leading to a successful immune response without actually moving to the nucleus. One of my major research focuses will be on elucidating the regulation and signaling of membrane-localized NLRs.
Recent work also indicates that several NLRs function in pairs or need so called “helper”-NLRs for their full activity. However, how these “helper”-NLRs function during an immune response or in autoimmunity and how specificity is encoded in such a signaling network is yet unknown. I want to understand the functional role “helper”- NLRs play in immunity (and autoimmunity). Up to now, we also lack knowledge about the importance of the host membrane trafficking system during immunity. My long-term research goal is to provide a mechanistic understanding of the molecular relationship between the host membrane trafficking system and the plant immune response initiated by NLRs (with a focus on the membrane localized ones). Thus, I will not only expand our knowledge of plant immunity and NLR biology, but also add practical potentials to engineer crop plants with a directed and robust resistance, thereby contributing to provide effective, environmentally friendly, and sustainable solutions for improving future agriculture.
Pathogen recognition first happens at the plasma membrane (PM) where receptor-like kinases/proteins perceive the presence of pathogen-derived molecules and initiate the first layer of immunity. To abrogate this first immune response pathogens inject effector proteins into the host cell. Some effectors intervene with the immune response directly at the PM by modifying components of the membrane-localized immune-signaling machinery (i.e. Chung et al., 2014, CHoM). During this arms-race plants have evolved membrane- (and cytosolic-localized) NLR proteins to detect effector presence/activity, thus ensuring activation of effector-triggered immunity at the center of the pathogen-host battle field – the plasma membrane. For a few NLRs nucleo-cytosolic shuttling, upon activation, is required for their full function and to interact with nuclear-localized transcriptional regulators to initiate a defense response. However, how membrane-localized NLRs induce the activation of an immune response without moving to the nucleus remains elusive. Thus, the identification of their precise pre-and post-activation localization, mechanisms of activation and signal transduction, as well as their intracellular trafficking, would improve our understanding on how these membrane-localized sensor NLRs eventually activate immunity. Recent work demonstrates that some NLRs function in pairs to initiate immunity, i.e. the NLR pairs RGA4/RGA5 in rice or RPS4/RRS1 in Arabidopsis. Other examples indicate that the activity of some (maybe all) NLRs during an immune response or in autoimmunity depends on the presence of members of a certain NLR subfamily, termed “helper”-NLRs. How exactly these “helper”-NLRs are regulated and how they translate the activation of “sensor/executer” NLRs needs to be investigated. Understanding how they get activated, what there signaling partners are and if they physically interact with sensor/executer NLRs is very important to understand and manipulate a plants immune system. Although it was shown that secretion of anti-microbial compounds, endocytosis of PAMP-recognition receptors and their vacuolar trafficking play a pivotal role in plant immunity, unexpectedly, only a few effectors from plant associated pathogens have been found to specifically target this trafficking system so far. Therefore, it is of great importance to identify effector targets and determine their subcellular localization to elucidate the complex mechanism of where and how the plants membrane trafficking system is manipulated in order to dampen plant immunity or to establish a beneficial interaction.
1. Biology of membrane localized NLR proteins.
The function of Arabidopsis plasma membrane localized NLRs RPM1, RPS2 and RPS5, the endosomal localized potato R3a (only the activated!), and the Golgi and tonoplast localized flux L6 and M, respectively, is well documented. However, little is known about how these and other membrane-localized sensor NLRs activate the nuclear reprogramming required for ETI, or whether these NLRs relocalize upon activation or what their downstream signaling partners are. To identify and characterize how membrane localized NLRs are regulated and how they signal, we will determine:
I. Where NLRs containing a conserved myristoylation and palmitoylation site localize
and if membrane localization is important for their function?
II. What are the regulators and signaling partners of membrane-localized NLRs?
2. Functional role of “helper”-NLRs in (auto-) immunity.
Only recently plant NLR functions were differentiated into “sensor”, “executer” or “helper” functions. Interestingly, neither plant nor animal “helper”-NLRs (i.e. ADR1-L2 and NLRC4, respectively) require a functional nucleotide-binding motif to fulfill their helper functions. In addition, the genomes of several plants, including rice, maize and Arabidopsis, encode for NLRs with degenerated nucleotide-binding motifs in their nucleotide-binding domain. Although, an important role of some of the “helper”-NLRs in plant immunity is described, no clear mechanistic understanding of their function exists yet. We will elucidate the following important issues:
I. What are the cellular components associated with “helper”-NLRs?
II. What are the molecular and genetic components of the autoimmunity
caused by an autoactive mutant “helper”- NLR?
III. How is the loss of “helper”-NLRs affecting a plants immune system and development
3. Importance of the membrane trafficking machinery in immunity.
Even though many trafficking pathways, like secretion, endocytosis or vacuolar transport, have been shown to be important for plant immunity, only a few trafficking components are known as having a direct function in immunity, i.e. the secretory SNARE complex formed by PEN1/SNAP33/VAMP721,722. Interestingly, some trafficking mutants, next to their trafficking defects, show (ectopic) activation of immunity commonly displayed in a dwarf stature, lesion formation on leaves and, in some cases, increased levels of the defense hormone salicylic acid. One such mutant, snap33, was so far completely neglected – probably due to its seedlings-lethality. We aim to identify suppressor mutants of the lesion formation in leaves of the snap33 mutant. Additionally, we will use the cell biological knowledge and skill set of the ZMBP to identify effector-targeted trafficking components and to elucidate how effectors manipulate their function during membrane trafficking. We propose to approach these issues by addressing the following questions:
I. What are the components regulating lesion formation in the snap33 mutant?
II. What effectors interact directly with trafficking components?
III. What is the subcellular localization of effectors inside the plant cell?
Through uncovering intracellular effector localization and their targets in the cellular trafficking machinery combined with the identification of how the membrane machinery is involved in ETI, mediated by membrane-localized NLR proteins, we hope that our research will greatly contribute to the endeavors in understanding plant-microbe interactions and plant immunity and thus directly contributes to improving agriculture.