Joseph F. McKenna

Cell Wall Damage-Induced Lignin Biosynthesis Is Regulated by a Reactive Oxygen Species- and Jasmonic Acid-Dependent Process in Arabidopsis

The plant cell wall is a dynamic and complex structure whose functional integrity is constantly being monitored and maintained during development and interactions with the environment. In response to cell wall damage (CWD), putatively compensatory responses, such as lignin production, are initiated. In this context, lignin deposition could reinforce the cell wall to maintain functional integrity. Lignin is important for the plant’s response to environmental stress, for reinforcement during secondary cell wall formation, and for long-distance water transport. Here, we identify two stages and several components of a genetic network that regulate CWD-induced lignin production in Arabidopsis (Arabidopsis thaliana). During the early stage, calcium and diphenyleneiodonium-sensitive reactive oxygen species (ROS) production are required to induce a secondary ROS burst and jasmonic acid (JA) accumulation. During the second stage, ROS derived from the NADPH oxidase RESPIRATORY BURST OXIDASE HOMOLOG D and JA-isoleucine generated by JASMONIC ACID RESISTANT1, form a negative feedback loop that can repress each other’s production. This feedback loop in turn seems to influence lignin accumulation. Our results characterize a genetic network enabling plants to regulate lignin biosynthesis in response to CWD through dynamic interactions between JA and ROS.

The Arabidopsis leucine-rich repeat receptor kinase MIK2/LRR-KISS connects cell wall integrity sensing, root growth and response to abiotic and biotic stresses

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues.

Chp8, a Diguanylate Cyclase from Pseudomonas syringae pv. Tomato DC3000, Suppresses the Pathogen-Associated Molecular Pattern Flagellin, Increases Extracellular Polysaccharides, and Promotes Plant Immune Evasion

ABSTRACT The bacterial plant pathogen Pseudomonas syringae causes disease in a wide range of plants. The associated decrease in crop yields results in economic losses and threatens global food security. Competition exists between the plant immune system and the pathogen, the basic principles of which can be applied to animal infection pathways. P. syringae uses a type III secretion system (T3SS) to deliver virulence factors into the plant that promote survival of the bacterium. The P. syringae T3SS is a product of the hypersensitive response and pathogenicity (hrp) and hypersensitive response and conserved (hrc) gene cluster, which is strictly controlled by the codependent enhancer-binding proteins HrpR and HrpS. Through a combination of bacterial gene regulation and phenotypic studies, plant infection assays, and plant hormone quantifications, we now report that Chp8 (i) is embedded in the Hrp regulon and expressed in response to plant signals and HrpRS, (ii) is a functional diguanylate cyclase, (iii) decreases the expression of the major pathogen-associated molecular pattern (PAMP) flagellin and increases extracellular polysaccharides (EPS), and (iv) impacts the salicylic acid/jasmonic acid hormonal immune response and disease progression. We propose that Chp8 expression dampens PAMP-triggered immunity during early plant infection. IMPORTANCE The global demand for food is projected to rise by 50% by 2030 and, as such, represents one of the major challenges of the 21st century, requiring improved crop management. Diseases caused by plant pathogens decrease crop yields, result in significant economic losses, and threaten global food security. Gaining mechanistic insights into the events at the plant-pathogen interface and employing this knowledge to make crops more resilient is one important strategy for improving crop management. Plant-pathogen interactions are characterized by the sophisticated interplay between plant immunity elicited upon pathogen recognition and immune evasion by the pathogen. Here, we identify Chp8 as a contributor to the major effort of the plant pathogen Pseudomonas syringae pv. tomato DC3000 to evade immune responses of the plant. The global demand for food is projected to rise by 50% by 2030 and, as such, represents one of the major challenges of the 21st century, requiring improved crop management. Diseases caused by plant pathogens decrease crop yields, result in significant economic losses, and threaten global food security. Gaining mechanistic insights into the events at the plant-pathogen interface and employing this knowledge to make crops more resilient is one important strategy for improving crop management. Plant-pathogen interactions are characterized by the sophisticated interplay between plant immunity elicited upon pathogen recognition and immune evasion by the pathogen. Here, we identify Chp8 as a contributor to the major effort of the plant pathogen Pseudomonas syringae pv. tomato DC3000 to evade immune responses of the plant.

Epidermal Pavement Cells of Arabidopsis Have Chloroplasts.

Plastids are multifunctional, pleomorphic organelles of purported endosymbiotic origin that in plants and green algae display a characteristic double membrane envelope ([Wise, 2007][1]). All plastids originate from colorless proplastids, and a simple pigmentation-based classification distinguishes

Across the great divide: the plant cell surface continuum.

The plant cell wall, plasma membrane and cytoskeleton exist as a cell surface continuum. This interconnection of organelles forms the interface between the plant cell and the external environment and is important for detecting the presence of a diverse range of stimuli. A plethora of plasma membrane microdomains with putative roles in membrane localized enzymatic or signalling processes have been described. While regulation of cell wall composition is defined by proteins within the plasma membrane, the cell wall has been shown to have an anchoring role on plasma membrane proteins which affects their lateral mobility. This interplay between plasma membrane and cell wall components is necessary for plasma membrane microdomain function. Actin and microtubule cytoskeletons are also involved in maintenance and function of the cell surface continuum. Investigation of the interactions between organellar components of this mechanism are important if we are to understand how cells respond to external signals.

Super-resolution nanoscopy imaging applied to DNA double strand breaks

Genomic deoxyribonucleic acid (DNA) is continuously being damaged by endogenous processes such as metabolism or by exogenous events such as radiation. The specific phosphorylation of histone H2AX on serine residue 139, described as γ-H2AX, is an excellent indicator or marker of DNA double-strand breaks (DSBs). The yield of γ-H2AX (foci) is shown to have some correlation with the dose of radiation or other DSB-causing agents. However, there is some discrepancy in the DNA DSB foci yield among imaging and other methods such as gel electrophoresis. Super-resolution imaging techniques are now becoming widely used as essential tools in biology and medicine, after a slow uptake of their development almost two decades ago. Here we compare several super-resolution techniques used to image and determine the amount and spatial distribution of γ-H2AX foci formation after X-ray irradiation: stimulated emission depletion (STED), ground-state depletion microscopy followed by individual molecule return (GSDIM), structured illumination microscopy (SIM), as well as an improved confocal, Airyscan and HyVolution 2. We show that by using these super-resolution imaging techniques with as low as 30-nm resolution, each focus may be further resolved, thus increasing the number of foci per radiation dose compared to standard microscopy. Furthermore, the DNA repair proteins 53BP1 (after low-LET irradiations) and Ku70/Ku80 (from laser microbeam irradiation) do not always yield a significantly increased number of foci when imaged by the super-resolution techniques, suggesting that γ-H2AX, 53PB1 and Ku70/80 repair proteins do not fully co-localize on the units of higher order chromatin structure.

Pattern-Triggered Immunity And Cell Wall Integrity Maintenance Jointly Modulate Plant Stress Responses

Plant cells are surrounded by walls, which must often meet opposing functional requirements during plant growth and defense. The cells meet them by modifying wall structure and composition in a tightly controlled and adaptive manner. The modifications seem to be mediated by a dedicated cell wall integrity (CWI) maintenance mechanism. Currently the mode of action of the mechanism is not understood and it is unclear how its activity is coordinated with established plant defense signaling. We investigated responses to induced cell wall damage (CWD) impairing CWI and the underlying mechanism in Arabidopsis thaliana. Interestingly inhibitor- and enzyme-derived CWD induced similar, turgor-sensitive stress responses. Genetic analysis showed that the receptor-like kinase (RLK) FEI2 and the mechano-sensitive, plasma membrane-localized Ca2+- channel MCA1 function downstream of the THE1 RLK in CWD perception. Phenotypic clustering with 27 genotypes identified a core group of RLKs and ion channels, required for activation of CWD responses. By contrast, the responses were repressed by pattern-triggered immune (PTI) signaling components including PEPR1 and 2, the receptors for the immune signaling peptide AtPep1. Interestingly AtPep1 application repressed CWD-induced phytohormone accumulation in a PEPR1/2-dependent manner. These results suggest that PTI suppresses CWD-induced defense responses through elicitor peptide-mediated signaling during defense response activation. If PTI is impaired, the suppression of CWD-induced responses is alleviated, thus compensating for defective PTI. Significance statement Stress resistance and plant growth determine food crop yield and efficiency of bioenergy production from ligno-cellulosic biomass. Plant cell walls are essential elements of the biological processes, therefore functional integrity of the cell walls must be maintained throughout. Here we investigate the plant cell wall integrity maintenance mechanism. We characterize its mode of action, identify essential signaling components and show that the AtPep1 signaling peptide apparently coordinates pattern triggered immunity (PTI) and cell wall integrity maintenance in plants. These results suggest how PTI and cell wall modification coordinately regulate biotic stress responses with plants possibly compensating for PTI impairment through enhanced activation of stress responses regulated by the CWI maintenance mechanism.All plant cells are surrounded by walls, which must often meet opposing functional requirements during plant growth and defense. The walls meet them by activating cell wall signaling processes to modify structure/composition in a controlled manner. Such adaptive changes have been summarily described as cell wall plasticity and identified as major obstacle to knowledge-based modification of lignocellulosic biomass. Plasticity requires activity of the cell wall integrity (CWI) maintenance mechanism. This mechanism monitors the functional integrity of the cell wall and initiates compensatory processes upon cell wall damage (CWD). To date, neither the mode of action of the CWI maintenance mechanism nor its role in immunity are understood. We investigated the mechanism in Arabidopsis thaliana and found that CWD caused by different means induced similar, turgor sensitive responses, suggesting similar principles underlie all CWD responses. Genetic analysis found that the receptor-like kinase (RLK) FEI2 and the plasma membrane channel MCA1 function downstream of the RLK THE1. Phenotypic clustering with 24 genotypes identified a core group of RLKs and ion channels required for activating CWD responses. By contrast, responses are repressed by pattern triggered immune signaling components including PEPR1 and 2, receptors for the plant immune signaling peptide AtPep1. AtPep1 application repressed CWD induced phytohormone production in wildtype, but not in pepr1/2 seedlings. These results suggest that pattern-triggered immunity suppresses responses triggered by the CWI maintenance mechanism through AtPep1 / PEPR1/2-dependent signaling. However, if pattern-triggered immunity is impaired, the repression of CWI regulated responses is removed thus compensating for immunity impairment.

Identification and characterization of conserved and divergent genes encoding the nuclear envelope LINC complex in maize (Zea mays L.).

The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex serves as an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm and functions to maintain nuclear shape and architecture, as well as regulates chromosome dynamics during mitosis and meiosis. Knowledge of LINC complex composition and function in the plant kingdom is relatively limited, especially in the monocots which include the cereal grains and other grass species. We identified and classified 22 genes encoding candidate LINC complex and associated proteins in maize through bioinformatic and biochemical approaches. Representative KASH candidates were functionally validated in one or more assays including nuclear envelope localization, native or heterologous co-immunoprecipitation with antisera for ZmSUN2, and fluorescence recovery after photobleaching. These findings support a summary working model of the entire maize LINC and associated proteins complex with components found to be conserved across eukaryotes, unique to plants, or highly divergent and grass-specific. This model contributes a new experimental system for the cell biology of the nuclear envelope and new opportunities for future studies of the LINC complex in a model crop species.The LINC (Linker of Nucleoskeleton to Cytoskeleton) complex is an essential multi-protein structure spanning the nuclear envelope. It connects the cytoplasm to the nucleoplasm, functions to maintain nuclear shape and architecture, and regulates chromosome dynamics during cell division. Knowledge of LINC complex composition and function in the plant kingdom is primarily limited to Arabidopsis, but critically missing from the evolutionarily distant monocots which include grasses, the most important agronomic crops worldwide. To fill this knowledge gap, we identified and characterized 22 maize genes, including a new grass-specific KASH gene family. Using bioinformatic, biochemical, and cell biological approaches, we provide evidence that representative KASH candidates localize to the nuclear periphery and interact with ZmSUN2 in vivo. FRAP experiments using domain-deletion constructs verified that this SUN-KASH interaction was dependent on the SUN but not the coiled-coil domain of ZmSUN2. A summary working model is proposed for the entire maize LINC complex encoded by conserved and divergent gene families. These findings expand our knowledge of the plant nuclear envelope in a model grass species, with implications for both basic and applied cellular research. SUMMARY STATEMENT Genes encoding maize candidates for the core LINC and associated complex proteins have been comprehensively identified with functional validation by one or more assays for several of the KASH genes.

Is Actin Filament Sliding Responsible for Endoplasmic Reticulum and Golgi Movement

In plants, the actin cytoskeleton and myosins are fundamental for normal dynamics of the endomembrane system and cytoplasmic streaming. We present evidence that this is in part due to myosin driven sliding of actin filaments within a bundle which generates the motive force required for cell dynamics in planta. Abstract In plants both the Golgi apparatus and the endoplasmic reticulum (ER) are highly motile. The Golgi apparatus, consisting of numerous Golgi stacks, is physically tethered to the surface of the motile ER membrane. Evidence is inconclusive as to whether there is a direct interaction between these organelles and the actin cytoskeleton, although there are reports of linker proteins between actin filaments and the ER membrane. Here, we use a combination of fluorescence recovery after photobleaching and a photoactivation strategy to investigate whether myosin driven actin filament sliding is a contributing factor in ER movement and thus Golgi motility. Utilising three different actin binding fluorescent protein constructs we show that recovery of fluorescence after photobleaching and loss of fluorescence after photoactivation is impeded by overexpression of a truncated myosin tail domain known to slow ER movement. We conclude that ER movement is in part mediated by myosin driven sliding of actin filaments within bundles linked to the ER membrane.