We and plants detect “danger” by recognizing microbe-associated molecules and launch complex immune responses to prevent infections. Our research interest is to understand the biochemical and molecular mechanisms underlying the dynamic plant-microbe interactions, and the signaling crosstalk that orchestrates plant responses to diverse extrinsic and intrinsic signals. Our ultimate goal is to understand how the complex signaling networks cooperate to activate plant defense responses and apply the knowledge gained from model plants to economically important crops to facilitate strategic development of disease resistant crops in agriculture.
1. Phosphorylation and ubiquitination of plant immune sensory complexes
Plants sense invading microorganisms by detection of conserved signatures, termed microbe-associated molecular patterns (MAMPs) through pattern recognition receptors (PRR) and mount pattern-triggered immunity (PTI). Immune sensory PRR complexes are frontlines of host defense systems. Plant immune senor FLS2, a cell-surface receptor-like kinase (RLK), recognizes bacterial flagellin and initiates immune signaling by association with BAK1, another RLK. FLS2/BAK1 complex-mediated immune system provides a paradigm for understanding the complex immune signaling networks and uncovering conservation of host immune mechanisms in different eukaryotes. Our studies revealed that an FLS2/BAK1-associated cytosolic kinase BIK1 is rapidly phosphorylated and released from FLS2/BAK1 receptor complex upon flagellin perception to propagate immune signaling (Lu et al., PNAS, 2010, Lin et al., PNAS, 2013). BIK1 is a non-receptor dual-specificity kinase and both tyrosine and serine/threonine kinase activities are required for its functions in plant immune signaling, suggesting that tyrosine phosphorylation cascade functions as a common regulatory mechanism controlling membrane-resident receptor signaling in plants and metazoans (Lin et al., PNAS, 2014). Upon receptor activation, down-regulation of immune signaling is instrumental for preventing excessive or prolonged activation of immune responses. Our work revealed two E3 ubiquitin ligases, PUB12 and PUB13, which interact with BAK1 and are recruited to FLS2/BAK1 complex for FLS2 ubiquitination/degradation, thereby down-regulating flagellin signaling (Lu et al., Science 2011). We are further characterizing the roles of protein phosphorylation and ubiquitination in the activation and attenuation of immune receptors and signaling with biochemical, proteomic, genetic and functional genomic approaches. In particular, we aim to characterize genome-wide ubiquitination dynamics in plant immune signaling, to understand how BIK1 activates diverse immune responses, and to fill in the gap of receptor complex and evolutionarily conserved MAP kinase cascade, a convergence downstream of multiple immune sensors.
2. Genetic dissection of plant immune gene expression and defense responses
Forward genetic screens have been proven powerful in various organisms for assigning gene functions and understanding signal transduction pathways in a myriad of biological processes. To explore additional PTI signaling components, we developed a sensitive genetic screen with an ethyl methanesulfonate (EMS)-mutagenized population of Arabidopsis transgenic plants carrying a luciferase reporter gene under the control of the FRK1 promoter (pFRK1::LUC). The FRK1 (flg22-induced receptor-like kinase 1) gene is a specific and early immune responsive gene activated by multiple MAMPs. A series of mutants with altered pFRK1::LUC activity upon flg22 treatment were identified and named as Arabidopsis genes governing immune gene expression (aggie). Molecular and biochemical analysis of Aggie1 led to the discovery of a phosphorylation circuit that regulates immune gene reprogramming via direct modulation of RNA polymerase II C-terminal domain (CTD) phosphorylation dynamics orchestrated by MAP kinases, CTD kinases and phosphatases. Characterization of Aggie2 indicates that protein poly(ADP-ribosyl)ation (PARylation) post-translational modification plays a critical role in plant immune gene expression and defense to pathogen attacks. PARylation is primarily mediated by poly(ADP-ribose) polymerase (PARP), which transfers ADP-ribose moieties from NAD+ to acceptor proteins. The covalently attached poly(ADP-ribose) polymers on the acceptor proteins could be hydrolyzed by poly(ADP-ribose) glycohydrolase (PARG). The coordinative action of PARPs and PARGs plays a crucial role in a broad array of cellular responses including DNA repair, cell division, chromatin modification and gene transcriptional regulation.
In addition, plants could recognize pathogen-encoded effectors by intracellular nucleotide-binding leucine-rich repeat (NLR) proteins to initiate effector-triggered immunity (ETI). Plant NLR resistance genes have been widely used in agriculture for improvement of resistance to a wide range of pathogens. However, the signaling regulators downstream of plant NLR resistance proteins remain elusive. We have developed a genetic screen with Arabidopsis transgenic plants carrying WRKY46 promoter fused with a luciferase reporter. As an early marker gene, WRKY46 is quickly and strongly activated by bacterial effectors triggering ETI, but not by MAMPs triggering PTI. Identification and characterization of ETI genes will likely reveal components and signaling mechanisms upon NLR activation.
3. Functional plasticity of shared signaling components in plant immunity, growth, cell differentiation and cell death.
Maintaining active growth and effective immune responses is often costly for a living organism to survive. Fine-tuning the shared modules is crucial for metazoans and plants to make a trade-off between growth and immunity. We reported that BIK1, a positive regulator in plant immunity, functions as a negative regulator in plant hormone brassinosteroid (BR)-mediated growth through association with BR receptor BRI1. The dynamic association of BIK1 with BAK1 and BRI1 may contribute to the inverse functions of BIK1 in plant immunity and development (Lin et al., PNAS 2013). BAK1 is involved in multiple biological processes, including plant immunity, development and cell death control via association with specific receptors. Thus, BAK1 serves as a shared signaling module that orchestrates the interconnected architecture of the complex cellular signaling networks yet disseminates diverse biological outcomes. We aim to use BIK1 and BAK1 as examples to understand how biological systems integrate diverse external and developmental cues to elicit specific biological outcomes with systems approaches and synthetic biology. In particular, we attempt to use optogenetic approach to selectively activate isolated modules in a specific pathway, which allows to track information flow to downstream specific responses with spatial and temporal controls in living cells.
4. Cotton functional genomics in biotic and abiotic stress responses
Cotton sustains one of the world’s largest industries (textiles) and serves as a significant source of fiber, feed, foodstuff, oil and biofuel products. However unraveling of cotton gene functions remains critically challenging as its painstaking transformation, limited tools for cotton gene manipulation and considerable size of genome. We have developed an Agrobacterium-mediated virus-induced gene silencing (VIGS) assay and a protoplast transient gain-of-function assay to dissect gene functions in cotton (Gao et al., Plant Journal, 2011, Gao et al., JIPB, 2014). These technique advances together with next generation sequencing allow us to dissect the molecular and biochemical regulatory mechanisms of cotton genes under different biotic and abiotic stresses. We have constructed a VIGS cotton cDNA library for the studies of cotton disease resistance and drought tolerance. In addition, in collaboration with Adam Bogdanove at Cornell University, we are using a combination of RNA-seq, bioinformatics prediction and molecular functional assays to identify cotton resistance and susceptibility genes to pathogenic bacteria Xanthomonas, which inject transcription activator-like (TAL) effector proteins into host cells to modulate host gene transcriptional reprograming. Our work will not only contribute to cotton basic research, but also provide genetic resources for improving cotton cultivars with sustainable disease resistance and drought tolerance in the field.
5. Evolution of plant immune signaling mechanisms
Metazoans and plants have evolved complex mechanisms to cope with the constant challenges of environmental stresses while maintaining their growth and development. Being sessile and lacking a sophisticated adaptive immune system, flowering plants have evolved a large number of RLKs, NLR proteins and MAP kinases and transcription factors that modulate growth, development and defense responses. It remains challenge to uncover the specific functions of individual key regulators that relay immune signaling from plasma membrane to nucleus. An ancient species with a significant reduced gene number of these multigene family will provide an ideal model to study their original function and evolution. Bioinformatic analysis has suggested that green algae Chlamydomonas reinhardtii only possesses one RLK gene and one WRKY gene, and the moss Physcomitrella patens has two NLR genes. We are using systems biology, comparative and functional genomics to understand the functions of these key regulators in lower plants and elucidate the origin and evolutionary history and functions of these key immune regulators.
Funding: NIH, NSF, USDA, Welch, Agrilife