Despite the ability to mount myriad immune responses, every plant or animal is still highly susceptible to numerous pathogens. Why? Answering this question is of fundamental importance in medicine and agriculture, as it holds a key to globally understanding infectious diseases in plants and humans. Ultimately, our goal is to answer two major questions:


                  1) How do microbial pathogens manipulate plants to cause disease?
            
2) How can we use pathogenesis as a probe for discovering fundamental cellular mechanisms in eukaryotic cells?





Our Model: Pseudomonas syringae - Arabidopsis thaliana




We use a model pathosystem consisting of the host Arabidopsis thaliana and the bacterial pathogen Pseudomonas syringae for our study. In this model interaction, both the host and the pathogen are genetically and genomically tractable, making it a powerful system in which to elucidate many of the basic principles that govern pathogenesis in eukaryotic hosts. To cause disease, P. syringae produce a variety of virulence factors, including dozens of "effector proteins" that are secreted through the type III secretion system (T3SS) and the phytotoxin coronatine, which functions as a molecular mimic of the plant hormone jasomonate. We have made steady progress in the understanding of how these virulence factors manipulate host innate immunity, vesicle trafficking, jasmonate signaling, and stomatal function.







Bacterial effector proteins: a central role in promoting disease susceptibility

Type-III secretion DC3000 Arabidopsis
Over the years, we have studied a number of different T3SS effectors, but our current efforts are mostly focused on two highly conserved effectors that are central to the ability of P. syringae and many other bacterial pathogens to cause disease in plants: HopM1 and AvrE. Our overarching goals in this research area are (1) to understand why HopM1 and AvrE make such a critical contribution to promoting disease susceptibility in diverse bacterial diseases, and (2) to inhibit these two effectors as a potential general strategy for bacterial disease control. We have identified the host targets of HopM1 in Arabidopsis. In particular, we have shown that HopM1 binds to the Arabidopsis ARF-GEF protein AtMIN7, a regulator of vesicle traffic (by activating the ARF family of GTPases) that is required for plant immune response. The physical interaction of HopM1 with MIN7 triggers the ubiquitination and subsequent degradation of MIN7 through the host proteasome. Our recent experiments show that both HopM1 and MIN7 are localized in trans-Golgi network (TGN)/endosome compartments. Remarkably, HopM1 also interacts with Rad23 proteins, which deliver ubiquitinated proteins to the proteasome. This finding leads us to suggest that HopM1 effector may hijack a putative endosome ubiquitination/proteasome system to degrade MIN7. We are attempting to identify additional components of the putative endosome-associated degradation machinery using HopM1 and MIN7 as probes in protein complex trapping and purification.



Plant stomata: the immune function against human and plant pathogens

DC3000 Stomata
Plant stomata are microscopic pores on the surface of all land plants; they are absolutely essential for exchange of CO2 gas and water vapor with the environment. As such, these pores are indispensable for plants to perform the most important function on earth: photosynthesis. In the plant pathology discipline, it has long been assumed that stomata serve as passive portals of entry for plant pathogens, particularly bacterial pathogens. However, our recent work shows that plant stomata have an important immune function. Specifically, stomata close in response to plant and human pathogenic bacteria. Stomatal guard cells could perceive bacteria and pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors, such as flagellin receptor FLS2, activating a signaling cascade that requires the plant stress hormones salicylic acid and abscisic acid.

A newly discovered immune response, the signal transduction pathway underlying stomatal closure to pathogens, is poorly characterized. We are taking several approaches to increase our understanding in this area. First, we are investigating the epistatic relationships between various signaling pathways in the stomatal guard cell. Second, we are isolating Arabidopsis mutants based on compromised stomatal response to P. syringae bacteria to identify new signaling components involved in the pathogen-triggered guard cell immune response. Third, because stomatal opening and closing are also regulated by abiotic signals, such as humidity, temperature, and CO2 concentration, we are studying potential cross-talk between stomatal responses to abiotic and biotic signals. This is particularly relevant to bacterial infections, as bacterial disease outbreaks often occur after rains and/or periods of high humidity. An exciting hypothesis is that stomatal immune response to pathogens may be compromised under disease-promoting weather conditions. Our ongoing experiments will test this hypothesis.





Coronatine: a remarkable molecular probe of the jasmonate receptor and signaling

Jasmonate signallingFor many years, we have been fascinated by a bacterial toxin called coronatine, because its chemical structure shares striking similarities to the plant hormone jasmonate. Jasmonate is a lipid-derived hormone that plays a crucial role in plant growth, development, and immunity. On this research topic, we collaborate with our colleague, Gregg Howe, who studies jasmonate signaling, especially in the context of wounding and insect interactions. We have used coronatine as a molecular probe in the identification of key regulators (e.g., JAZ repressors) and components of the jasmonate receptor complex. In these studies we also collaborate with the laboratories of John Browse at Washington State University and Ning Zheng at the University of Washington.

The identification of the JAZ repressor proteins (a total of twelve in Arabidopsis) is unleashing a wave of studies to define the entire JA regulon and many roles of JA signaling in development, growth, and immunity. We will continue to focus our attention on the role of coronatine/jasmonate signaling in plant-pathogen interactions, because this topic hasbeen traditionally understudied and has particularly high potential for new discoveries. Jasmonate signaling has opposite effects on pathogens of different lifestyles: it promotes infection of biotrophic pathogens (which multiply in living host tissues), but inhibits infection of necrotrophic pathogens (which live in dead host tissues). The molecular bases of these opposing effects are not known and remain a fundamental question. Our overall hypothesis is that different JAZ repressors interact with common as well as unique downstream transcription factors to specify common and varied downstream outputs based on specific external stimuli.






Recent Research Initiatives: The Next Phase of Study

Despite significant advances, current understanding of disease susceptibility in plants remains largely one-dimensional, reflecting the heavy reliance on simplistic bilateral interactions of one pathogen and one host under static laboratory conditions. As a result, our knowledge of disease susceptibility does not accurately reflect the multi-dimensional features of plant disease development that occur in nature. To break new ground for the next phase of research on plant disease susceptibility, we have initiated the following new research projects.


Basic principals of future study for the He lab






Environment and disease: understanding the molecular basis of the “disease triangle” dogma

Plant disease triangle

A long-standing dogma in plant disease susceptibility states that disease development requires not only the presence of a virulent pathogen and a susceptible host, but also a set of disease-favoring environmental conditions. How environment conditions influence the plant and the pathogen during an active interaction is poorly understood, leaving a big gap in our understanding of how disease outbreaks occur in nature. To gain insight into the molecular basis of the “disease triangle” dogma, we initiated a project aimed at elucidating how two prominent abiotic environmental factors (temperature and humidity) intercept the molecular network associated with disease development.









Plant microbiome and disease: development of a soil-based gnotobiotic system for plant research


Plant Microbiome and Disease
Current studies of disease susceptibility in plants have largely ignored potential effects of the microbiome that is indigenous to each plant. Anticipating that the next phase of research on disease susceptibility needs to consider the microbiome as an integral component of a multi-dimensional interaction during disease development, we initiated a project to develop a soil-based gnotobiotic plant growth system (called “FlowPot”). Similar to the “germ-free mouse” gnotobiotic system for medical research, we hope that the FlowPot system would enable plant scientists to study the role of the microbiome in modulating various aspects of plant biology.






Application of our basic research: inactivation of pathogen virulence systems

Coronatine Receptor Coi1In the past decade, characterization of host targets of pathogen virulence factors took center stage in the study of pathogenesis and disease susceptibility in plants and humans. However, the impressive knowledge of host targets has not been broadly exploited to inhibit pathogen infection. We initiated efforts to explore host target modification as a promising new approach to “protect” disease-vulnerable components of plant. Recently, we succeeded in modifying the jasmonate hormone receptor to greatly reduce sensitivity to coronatine, a potent virulence factor that mimics jasmonate and hijacks the jasmonate receptor. Our results provide a proof-of-concept demonstration that host target modification can be a promising new approach to prevent virulence action of highly evolved pathogens.

In addition, due to the central role the type III protein secretion apparatus plays in causing bacterial infections in plants and humans, there have been various efforts to inactivate this apparatus as a strategy for bacterial disease control. However, currently very little is known of the molecular interplay between the type III secretion apparatus and plant defense. We are investigating whether there is natural plant defense against the type III secretion apparatus. If successful, we may be able to develop innovative plant defense-based approaches to block this central virulence apparatus conserved in diverse bacterial pathogens.


Title image: A dual column FIB scanning electron micrograph image of the Arabidopsis leaf surface. The Spartan helmet logo and "Sheng Yang He Lab" were etched directly onto an Arabidopsis leaf using an focused ion beam (FIB) mill.