Sarah R. Hind

Allelic variation in two distinct Pseudomonas syringae flagellin epitopes modulates the strength of plant immune responses but not bacterial motility

The bacterial flagellin (FliC) epitopes flg22 and flgII-28 are microbe-associated molecular patterns (MAMPs). Although flg22 is recognized by many plant species via the pattern recognition receptor FLS2, neither the flgII-28 receptor nor the extent of flgII-28 recognition by different plant families is known. Here, we tested the significance of flgII-28 as a MAMP and the importance of allelic diversity in flg22 and flgII-28 in plant-pathogen interactions using purified peptides and a Pseudomonas syringae ∆fliC mutant complemented with different fliC alleles. The plant genotype and allelic diversity in flg22 and flgII-28 were found to significantly affect the plant immune response, but not bacterial motility. The recognition of flgII-28 is restricted to a number of solanaceous species. Although the flgII-28 peptide does not trigger any immune response in Arabidopsis, mutations in both flg22 and flgII-28 have FLS2-dependent effects on virulence. However, the expression of a tomato allele of FLS2 does not confer to Nicotiana benthamiana the ability to detect flgII-28, and tomato plants silenced for FLS2 are not altered in flgII-28 recognition. Therefore, MAMP diversification is an effective pathogen virulence strategy, and flgII-28 appears to be perceived by an as yet unidentified receptor in the Solanaceae, although it has an FLS2-dependent virulence effect in Arabidopsis.

Tomato receptor FLAGELLIN-SENSING 3 binds flgII-28 and activates the plant immune system

Plants and animals detect the presence of potential pathogens through the perception of conserved microbial patterns by cell surface receptors. Certain solanaceous plants, including tomato, potato and pepper, detect flgII-28, a region of bacterial flagellin that is distinct from that perceived by the well-characterized FLAGELLIN-SENSING 2 receptor. Here we identify and characterize the receptor responsible for this recognition in tomato, called FLAGELLIN-SENSING 3. This receptor binds flgII-28 and enhances immune responses leading to a reduction in bacterial colonization of leaf tissues. Further characterization of FLS3 and its signalling pathway could provide new insights into the plant immune system and transfer of the receptor to other crop plants offers the potential of enhancing resistance to bacterial pathogens that have evolved to evade FLS2-mediated immunity.

Tissue-type specific systemin perception and the elusive systemin receptor.

Systemin is a wound signaling peptide from tomato that is important for plant defenses against herbivory. The systemin receptor was initially identified as the tomato homolog of the brassinosteroid receptor BRI1, but genetic evidence argued against this finding. However, we found that BRI1 may function as an inappropriate systemin binding protein that does not activate the systemin signaling pathway. Here we provide evidence that systemin perception is localized in a tissue-type specific manner. Mesophyll protoplasts were not sensitive to systemin, while they responded to other elicitors. We hypothesize that the elusive systemin receptor is a protein with high similarity to BRI1 which is specifically localized in vascular tissue like the systemin precursor prosystemin. Binding of systemin to BRI1 may be an artifact of transgenic BRI1-overexpressing plants, but does not take place in wild type tomato cells.

Natural variation for responsiveness to flg22, flgII-28, and csp22 and Pseudomonas syringae pv. tomato in heirloom tomatoes

Tomato (Solanum lycopersicum L.) is susceptible to many diseases including bacterial speck caused by Pseudomonas syringae pv. tomato. Bacterial speck disease is a serious problem worldwide in tomato production areas where moist conditions and cool temperatures occur. To enhance breeding of speck resistant fresh-market tomato cultivars we identified a race 0 field isolate, NC-C3, of P. s. pv. tomato in North Carolina and used it to screen a collection of heirloom tomato lines for speck resistance in the field. We observed statistically significant variation among the heirloom tomatoes for their response to P. s. pv. tomato NC-C3 with two lines showing resistance approaching a cultivar that expresses the Pto resistance gene, although none of the heirloom lines have Pto. Using an assay that measures microbe-associated molecular pattern (MAMP)-induced production of reactive oxygen species (ROS), we investigated whether the heirloom lines showed differential responsiveness to three bacterial-derived peptide MAMPs: flg22 and flgII-28 (from flagellin) and csp22 (from cold shock protein). Significant differences were observed for MAMP responsiveness among the lines, although these differences did not correlate strongly with resistance or susceptibility to bacterial speck disease. The identification of natural variation for MAMP responsiveness opens up the possibility of using a genetic approach to identify the underlying loci and to facilitate breeding of cultivars with enhanced disease resistance. Towards this goal, we discovered that responsiveness to csp22 segregates as a single locus in an F2 population of tomato.

Gene silencing goes viral and uncovers the private life of plants

To understand how genes influence plant responses to the environment, it is important to have appropriate tools for studying gene function. This may be a challenge for non‐model plant species or plants that are recalcitrant to genetic transformation. Virus‐induced gene silencing (VIGS) has been established as a convenient, rapid, and efficient method for reverse genetics in a wide range of plant species ranging from non‐model dicot plants to cereal crop plants. Virus‐induced gene silencing is not limited to the green parts of a plant; genes can also be efficiently silenced in roots and reproductive organs. As it is established during later developmental stages, it avoids the problem of embryo‐ or seedling lethality, which may impede the investigation of some null mutants or transgenic knock‐out plants. Virus‐induced gene silencing can be designed to target specific members of a gene family, or to co‐silence paralogous genes, and it has been adapted for high‐throughput analysis. Virus‐induced gene silencing has frequently been used to study plant–pathogen interactions and plant development. This review emphasizes the great potential of VIGS for the study of plant–arthropod interactions, ranging from plant–herbivore and plant–pollinator interactions to behavioral animal responses to plants. In spite of this potential, VIGS has not often been adopted by entomologists and plant scientists who study plant–arthropod interactions. We also provide practical considerations for developing or adopting a VIGS system.

Wounding systemically activates a mitogen-activated protein kinase in forage and turf grasses

Forage and turf grasses are continually cut and grazed by livestock, however very little is known concerning the perception or molecular responses to wounding. Mechanical wounding rapidly activated a 46 kDa and a 44 kDa mitogen-activated protein kinase (MAPK) in six different grass species. In the model grass species Lolium temulentum, the 46 kDa MAPK was rapidly activated within 5 min of wounding both locally and systemically in an adjacent unwounded tiller. This indicates that wounding generates a rapidly propagated long-distance signal that activates a MAPK in the distal portions of the plant. This 46 kDa MAPK activity was not enhanced by the addition of the pathogen-associated signal salicylic acid (SA) to the wound site nor induced when exposed to methyl jasmonate (MJ), which is a potent inducer of the wound response in dicotyledonous plants. However, pretreatment with MJ increased the wound-induced activity of the 44 kDa MAPK over the activity in control plants.

Detecting N-myristoylation and S-acylation of host and pathogen proteins in plants using click chemistry.

BackgroundThe plant plasma membrane is a key battleground in the war between plants and their pathogens. Plants detect the presence of pathogens at the plasma membrane using sensor proteins, many of which are targeted to this lipophilic locale by way of fatty acid modifications. Pathogens secrete effector proteins into the plant cell to suppress the plant’s defense mechanisms. These effectors are able to access and interfere with the surveillance machinery at the plant plasma membrane by hijacking the host’s fatty acylation apparatus. Despite the important involvement of protein fatty acylation in both plant immunity and pathogen virulence mechanisms, relatively little is known about the role of this modification during plant-pathogen interactions. This dearth in our understanding is due largely to the lack of methods to monitor protein fatty acid modifications in the plant cell.ResultsWe describe a rapid method to detect two major forms of fatty acylation, N-myristoylation and S-acylation, of candidate proteins using alkyne fatty acid analogs coupled with click chemistry. We applied our approach to confirm and decisively demonstrate that the archetypal pattern recognition receptor FLS2, the well-characterized pathogen effector AvrPto, and one of the best-studied intracellular resistance proteins, Pto, all undergo plant-mediated fatty acylation. In addition to providing a means to readily determine fatty acylation, particularly myristoylation, of candidate proteins, this method is amenable to a variety of expression systems. We demonstrate this using both Arabidopsis protoplasts and stable transgenic Arabidopsis plants and we leverage Agrobacterium-mediated transient expression in Nicotiana benthamiana leaves as a means for high-throughput evaluation of candidate proteins.ConclusionsProtein fatty acylation is a targeting tactic employed by both plants and their pathogens. The metabolic labeling approach leveraging alkyne fatty acid analogs and click chemistry described here has the potential to provide mechanistic details of the molecular tactics used at the host plasma membrane in the battle between plants and pathogens.

MAP kinases associate with high molecular weight multiprotein complexes

Depending on their phosphorylation status, MAP kinases (MAPKs) dynamically associate with stable high molecular weight multiprotein complexes.

Detecting the Interaction of Peptide Ligands with Plant Membrane Receptors

The field of plant receptor biology has rapidly expanded in recent years, however the demonstration of direct interaction between receptor-ligand pairs remains a challenge. Click chemistry has revolutionized small molecule research but lacks popularity in plant research. Here we describe a method that tests for the direct physical interaction of a candidate receptor protein and a peptide ligand. This protocol describes the generation of the ligand probe, transient expression of a receptor protein, enrichment of membrane-bound receptors, photo-crosslinking and click chemistry-mediated reporter addition, and detection of the receptor-ligand complex. Copper-based click chemistry confers several advantages, including the versatility to use almost any azide-containing reporter molecule for detection or visualization of the complex and addition of the reporter molecule after receptor-ligand binding which reduces the need for bulky ligand modifications that could interfere with the interaction.