Measuring Pectin Properties to Track Cell Wall Alterations During Plant-Pathogen Interactions.

Plant cell walls act both as a barrier to pathogen entry and as a source of signaling molecules that can modulate plant immunity. Cell walls consist mainly of three polymeric sugars: cellulose, pectin, and hemicellulose (Mohnen et al., Biomass Recalcitrance: deconstructing the plant cell wall for bioenergy, 2008). In Arabidopsis more than 50% of the primary cell wall is pectin (Zablackis et al., Plant Physiol 107:1129-1138, 1995). There are various types of pectin, but all pectins contain galacturonic acid subunits in their backbone (Harholt et al., Plant Physiol 153:384-395, 2010; Mohnen, Curr Opin Plant Biol 11:266-277, 2008). Many pathogens secrete pectin-degrading enzymes as part of their infection strategy (Espino et al., Proteomics 10:3020-3034, 2010; ten Have et al., Mol Plant-Microbe Interact 11:1009-1016, 1998). Pectin is synthesized in a highly esterified fashion and is de-esterified in the cell wall by pectin methylesterases (Harholt et al., Plant Physiol 153:384-395, 2010; Mohnen, Curr Opin Plant Biol 11:266-277, 2008). During plant-pathogen interactions, both the amount and the patterns of pectin methylesterification in the wall can be altered (Bethke et al., Plant Physiol 164:1093-1107, 2014; Lionetti et al., J Plant Physiol 169:1623-1630, 2012). Pectin methylesterifications influence mechanical properties of pectin, and pectins must be at least partially de-methylesterified to be substrates for pectin-degrading enzymes (Levesque-Tremblay et al., Planta 242:791-811, 2015). Additionally, alterations of pectin methylesterification or pectin content affect pathogen growth (Bethke et al., Plant Physiol 164:1093-1107, 2014; Lionetti et al., J Plant Physiol 169:1623-1630, 2012; Bethke et al., Plant Cell 28:537-556, 2016; Raiola et al., Mol Plant-Microbe Interact 24:432-440, 2011; Vogel et al., Plant Cell 14:2095-2106, 2002; Vogel et al., Plant J 40:968-978, 2004; Wietholter et al., Mol Plant-Microbe Interact 16:945-952, 2003). This chapter explains a simple protocol that can be used in any molecular biology laboratory to estimate total pectin content using a colorimetric assay and pectin composition using antibodies raised against specific pectin components.

Different Modes of Negative Regulation of Plant Immunity by Calmodulin-Related Genes.

Plant immune responses activated through the perception of microbe-associated molecular patterns, leading to pattern-triggered immunity, are tightly regulated. This results in low immune responses in the absence of pathogens and a rapid return to the resting state following an activation event. Here, we show that two CALMODULIN-LIKE genes, CML46 and CML47, negatively regulate salicylic acid accumulation and immunity in Arabidopsis (Arabidopsis thaliana). The double mutant cml46 cml47 is highly resistant to the pathogen Pseudomonas syringae pv maculicola (Pma). The effects of cml46 cml47 on Pma growth are genetically additive to that of cbp60a, a known negative regulator in the CALMODULIN-BINDING PROTEIN60 (CBP60) family. Transcriptome profiling revealed the effects of cbp60a and cml46 cml47 on both common and separate sets of genes, with the majorities of these differentially expressed genes being Pma responsive. CBP60g, a positive regulator of immunity in the CBP60 family, was found to be transcriptionally regulated by CBP60a, CML46, and CML47 Analysis of the flg22-induced mRNA levels of CBP60g in cbp60a and cml46 cml47 revealed that cml46 cml47 plants have higher induced expression while cbp60a plants retain elevated levels longer than wild-type plants. Assays for the effect of flg22 treatment on Pma growth showed that the effect is stronger in cml46 cml47 plants and lasts longer in cbp60a plants. Thus, the expression pattern of CBP60g is reflected in flg22-induced resistance to Pma.

Comparative Genomic Analyses of Clavibacter michiganensis subsp. insidiosus and Pathogenicity on Medicago truncatula.

Clavibacter michiganensis is the most economically important gram-positive bacterial plant pathogen, with subspecies that cause serious diseases of maize, wheat, tomato, potato, and alfalfa. Much less is known about pathogenesis involving gram-positive plant pathogens than is known for gram-negative bacteria. Comparative genome analyses of C. michiganensis subspecies affecting tomato, potato, and maize have provided insights on pathogenicity. In this study, we identified strains of C. michiganensis subsp. insidiosus with contrasting pathogenicity on three accessions of the model legume Medicago truncatula. We generated complete genome sequences for two strains and compared these to a previously sequenced strain and genome sequences of four other subspecies. The three C. michiganensis subsp. insidiosus strains varied in gene content due to genome rearrangements, most likely facilitated by insertion elements, and plasmid number, which varied from one to three depending on strain. The core C. michiganensis genome consisted of 1,917 genes, with 379 genes unique to C. michiganensis subsp. insidiosus. An operon for synthesis of the extracellular blue pigment indigoidine, enzymes for pectin degradation, and an operon for inositol metabolism are among the unique features. Secreted serine proteases belonging to both the pat-1 and ppa families were present but highly diverged from those in other subspecies.

WRKY70 prevents axenic activation of plant immunity by direct repression of SARD1.

SARD1 is an activator of plant immunity that promotes production of the hormone salicylic acid (SA) and activation of defense gene expression. SARD1 itself is strongly inducible by infection. Here, we investigated the transcriptional control of SARD1. We used yeast one-hybrid assays to identify WRKY70. The WRKY70 binding site was defined using electrophoretic mobility shift assays, and its importance was investigated using an Arabidopsis thaliana protoplast system. The effect of wrky70 mutations was studied by measurements of pathogen growth, SA concentrations, and gene expression by RNA-seq. WRKY70 binds to a GACTTTT motif in the SARD1 promoter in yeast and Arabidopsis protoplasts. Plants with wrky70 mutations have elevated expression of SARD1 in the absence of pathogens, but not when infected. Expression profiling revealed that WRKY70 represses many pathogen-inducible genes in the absence of pathogens, yet is required for activation of many other pathogen-inducible genes in infected plants. The GACTTTT motif is enriched in the promoters of both these gene sets, and conserved in SARD1 orthologs within the Brassicaceae. WRKY70 represses SARD1 by binding the motif GACTTTT in the absence of pathogens. Conservation of the WRKY70 binding among the Brassicaceae suggests that WRKY70 repression of SARD1 is important for fitness.

A plant effector-triggered immunity signaling sector is inhibited by pattern-triggered immunity.

Since signaling machineries for two modes of plant-induced immunity, pattern-triggered immunity (PTI) and effector-triggered immunity (ETI), extensively overlap, PTI and ETI signaling likely interact. In an Arabidopsis quadruple mutant, in which four major sectors of the signaling network, jasmonate, ethylene, PAD4, and salicylate, are disabled, the hypersensitive response (HR) typical of ETI is abolished when the Pseudomonas syringae effector AvrRpt2 is bacterially delivered but is intact when AvrRpt2 is directly expressed in planta These observations led us to discovery of a network-buffered signaling mechanism that mediates HR signaling and is strongly inhibited by PTI signaling. We named this mechanism the ETI-Mediating and PTI-Inhibited Sector (EMPIS). The signaling kinetics of EMPIS explain apparently different plant genetic requirements for ETI triggered by different effectors without postulating different signaling machineries. The properties of EMPIS suggest that information about efficacy of the early immune response is fed back to the immune signaling network, modulating its activity and limiting the fitness cost of unnecessary immune responses.

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana.

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-D-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-D-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions.

Identification of differentially expressed genes between developing seeds of different soybean cultivars.

Soybean is a major source of protein and oil and a primary feedstock for biodiesel production. Research on soybean seed composition and yield has revealed that protein, oil and yield are controlled quantitatively and quantitative trait loci (QTL) have been identified for each of these traits. However, very limited information is available regarding the genetic mechanisms controlling seed composition and yield. To help address this deficiency, we used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean cultivars, which differ in seed weight, yield, protein content and oil content. A total of 700 probe sets were found to be expressed at significantly different (defined as having an adjusted p-value below or equal to 0.05 and an at least 2-fold difference) levels between the two cultivars at one or more of the three developmental stages and in at least one of the two years assayed. Comparison of data from soybeans collected in two different years revealed that 97 probe sets were expressed at significantly different levels in both years. Functional annotations were assigned to 78% of these 97 probe sets based on the SoyBase Affymetrix™ GeneChip® Soybean Genome Array Annotation. Genes involved in receptor binding/activity and protein binding are overrepresented among the group of 97 probe sets that were differentially expressed in both years assayed. Probe sets involved in growth/development, signal transduction, transcription, defense/stress response and protein and lipid metabolism were also identified among the 97 probe sets and their possible implications in the regulation of agronomic traits are discussed. As the Minsoy and Archer soybean cultivars differ with respect to seed size, yield, protein content and lipid content, some of the differentially expressed probe sets identified in this study may thus play important roles in controlling these traits. Others of these probe sets may be involved in regulation of general seed development or metabolism. All microarray data and expression values after GCRMA are available at the Gene Expression Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo), under accession number GSE21598.

Functional characterization of PCRK1, a putative protein kinase with a role in immunity.

In Arabidopsis, defense signaling is triggered by the perception of conserved molecular patterns by pattern recognition receptors (PRRs). Signal transduction from the PRRs requires members of a family of Receptor-Like Cytoplasmic Kinases (RLCKs). Previously, we described one such RLCK, PTI Compromised Receptor-Like Cytoplasmic Kinase 1 (PCRK1) that is important for immunity induced by Microbe Associated Molecular Patterns (MAMPs) as well as Damage Associated Molecular Patterns (DAMPs). In this study, we measured the growth of Pma ES4326 in double mutants carrying pcrk1 together with the salicylic acid (SA) biosynthesis mutation sid2-2 or the jasmonic acid (JA) receptor mutation coi1-1, showing that the function of PCRK1 is SA independent but may be partially dependent on JA. Mutation of phosphorylated serine residues S232, S233 and S237 compromised the immune signaling function of PCRK1.

Reassess the t Test: Interact with All Your Data via ANOVA.

Plant biology is rapidly entering an era where we have the ability to conduct intricate studies that investigate how a plant interacts with the entirety of its environment. This requires complex, large studies to measure how plant genotypes simultaneously interact with a diverse array of environmental stimuli. Successful interpretation of the results from these studies requires us to transition away from the traditional standard of conducting an array of pairwise t tests toward more general linear modeling structures, such as those provided by the extendable ANOVA framework. In this Perspective, we present arguments for making this transition and illustrate how it will help to avoid incorrect conclusions in factorial interaction studies (genotype × genotype, genotype × treatment, and treatment × treatment, or higher levels of interaction) that are becoming more prevalent in this new era of plant biology.

Putative Serine Protease Effectors of Clavibacter michiganensis Induce a Hypersensitive Response in the Apoplast of Nicotiana Species.

Clavibacter michiganensis subspp. michiganensis and sepedonicus cause diseases on solanaceous crops. The genomes of both subspecies encode members of the pat-1 family of putative serine proteases known to function in virulence on host plants and induction of hypersensitive responses (HR) on nonhosts. One gene of this family in C. michiganensis subsp. sepedonicus, chp-7, is required for triggering HR in Nicotiana tabacum. Here, further investigation revealed that mutation of the putative catalytic serine residue at position 232 to threonine abolished the HR induction activity of Chp-7, suggesting that enzymatic activity is required. Purified Chp-7 triggered an HR in N. tabacum leaves in the absence of the pathogen, indicating Chp-7 itself is the HR elicitor from C. michiganensis subsp. sepedonicus. Ectopic expression of chp-7 constructs in N. tabacum leaves revealed that Chp-7 targeted to the apoplast triggered an HR while cytoplasmic Chp-7 did not, indicating that Chp-7 induces the HR in the apoplast of N. tabacum leaves. Chp-7 also induced HR in N. sylvestris, a progenitor of N. tabacum, but not in other Nicotiana species tested. ChpG, a related protein from C. michiganensis subsp. michiganensis, also triggered HR in N. tabacum and N. sylvestris. Unlike Chp-7, ChpG triggered HR in N. clevelandii and N. glutinosa.

Complete Genome Sequence of Clavibacter michiganensis subsp. insidiosus R1-1 Using PacBio Single-Molecule Real-Time Technology.

We report here the complete genome sequence of Clavibacter michiganensis subsp. insidiosus R1-1, isolated in Minnesota, USA. The R1-1 genome, generated by a de novo assembly of PacBio sequencing data, is the first complete genome sequence available for this subspecies.

The receptor-like cytoplasmic kinase PCRK1 contributes to pattern-triggered immunity against Pseudomonas syringae in Arabidopsis thaliana.

In this paper we describe PATTERN-TRIGGERED IMMUNITY (PTI) COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) of Arabidopsis thaliana, an RLCK that is important for defense against the pathogen Pseudomonas syringae pv. maculicola ES4326 (Pma ES4326). We examined defense responses such as bacterial growth, production of reactive oxygen species (ROS) and callose deposition in pcrk1 mutant plants to determine the role of PCRK1 during pathogen infection. Expression of PCRK1 was induced following pathogen infection. Pathogen growth was significantly higher in pcrk1 mutant lines than in wild-type Col-0. Mutant pcrk1 plants showed reduced pattern-triggered immunity (PTI) against Pma ES4326 after pretreatment with peptides derived from flagellin (flg22), elongation factor-Tu (elf18), or an endogenous protein (pep1). Deposition of callose was reduced in pcrk1 plants, indicating a role of PCRK1 in activation of early immune responses. A PCRK1 transgene containing a mutation in a conserved lysine residue important for phosphorylation activity of kinases (K118E) failed to complement a pcrk1 mutant for the Pma ES4326 growth phenotype. Our study shows that PCRK1 plays an important role during PTI and that a conserved lysine residue in the putative kinase domain is important for PCRK1 function.

The mRNA decay factor PAT1 functions in a pathway including MAP kinase 4 and immune receptor SUMM2.

Multi-layered defense responses are activated in plants upon recognition of invading pathogens. Transmembrane receptors recognize conserved pathogen-associated molecular patterns (PAMPs) and activate MAP kinase cascades, which regulate changes in gene expression to produce appropriate immune responses. For example, Arabidopsis MAP kinase 4 (MPK4) regulates the expression of a subset of defense genes via at least one WRKY transcription factor. We report here that MPK4 is found in complexes in vivo with PAT1, a component of the mRNA decapping machinery. PAT1 is also phosphorylated by MPK4 and, upon flagellin PAMP treatment, PAT1 accumulates and localizes to cytoplasmic processing (P) bodies which are sites for mRNA decay. Pat1 mutants exhibit dwarfism and de-repressed immunity dependent on the immune receptor SUMM2. Since mRNA decapping is a critical step in mRNA turnover, linking MPK4 to mRNA decay via PAT1 provides another mechanism by which MPK4 may rapidly instigate immune responses.

Arabidopsis PECTIN METHYLESTERASEs contribute to immunity against Pseudomonas syringae.

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification.

Are skin scar characteristics associated with the degree of pelvic adhesions at laparoscopy?

OBJECTIVE: To investigate whether individual or a combination of abdominal surgical scar characteristics can predict the severity and extent of intra-abdominal adhesions. DESIGN: A prospective cohort study. SETTING: A tertiary referral center in the United Kingdom. PATIENT(S): One hundred women who had previously undergone abdominopelvic surgery and were undergoing an elective laparoscopic gynecologic operations. INTERVENTION(S): Abdominal scars were evaluated preoperatively using the modified Manchester Scar Questionnaire Adhesions were assessed intraoperatively and compared with the cutaneous findings. MAIN OUTCOME MEASURE(S): Presence and severity of intra-abdominal adhesions. RESULT(S): Of 100 women recruited into this study, 71 (71%) women were found to have intra-abdominal Aadhesions, and 29 (29%) had no adhesions. Women who had more than one abdominal scar, a palpable scar, and/or a longer scar were most likely to have pelvic adhesions during the current surgery. Women with the highest mean scar scores also had a greater total adhesion score. CONCLUSION(S): Adhesions are a common postoperative consequence of open or laparoscopic surgery. Skin scar characteristics are associated with the presence and degree of pelvic adhesions. Future studies should examine whether these characteristics can be used as a preoperative predictive tool to facilitate surgical decision-making and elective operating room organization.

Dual regulation of gene expression mediated by extended MAPK activation and salicylic acid contributes to robust innate immunity in Arabidopsis thaliana.

Network robustness is a crucial property of the plant immune signaling network because pathogens are under a strong selection pressure to perturb plant network components to dampen plant immune responses. Nevertheless, modulation of network robustness is an area of network biology that has rarely been explored. While two modes of plant immunity, Effector-Triggered Immunity (ETI) and Pattern-Triggered Immunity (PTI), extensively share signaling machinery, the network output is much more robust against perturbations during ETI than PTI, suggesting modulation of network robustness. Here, we report a molecular mechanism underlying the modulation of the network robustness in Arabidopsis thaliana. The salicylic acid (SA) signaling sector regulates a major portion of the plant immune response and is important in immunity against biotrophic and hemibiotrophic pathogens. In Arabidopsis, SA signaling was required for the proper regulation of the vast majority of SA-responsive genes during PTI. However, during ETI, regulation of most SA-responsive genes, including the canonical SA marker gene PR1, could be controlled by SA-independent mechanisms as well as by SA. The activation of the two immune-related MAPKs, MPK3 and MPK6, persisted for several hours during ETI but less than one hour during PTI. Sustained MAPK activation was sufficient to confer SA-independent regulation of most SA-responsive genes. Furthermore, the MPK3 and SA signaling sectors were compensatory to each other for inhibition of bacterial growth as well as for PR1 expression during ETI. These results indicate that the duration of the MAPK activation is a critical determinant for modulation of robustness of the immune signaling network. Our findings with the plant immune signaling network imply that the robustness level of a biological network can be modulated by the activities of network components.

The CALMODULIN-BINDING PROTEIN60 family includes both negative and positive regulators of plant immunity.

Two members of the eight-member CALMODULIN-BINDING PROTEIN60 (CBP60) gene family, CBP60g and SYSTEMIC ACQUIRED RESISTANCE DEFICIENT1 (SARD1), encode positive regulators of plant immunity that promote the production of salicylic acid (SA) and affect the expression of SA-dependent and SA-independent defense genes. Here, we investigated the other six family members in Arabidopsis (Arabidopsis thaliana). Only cbp60a mutations affected growth of the bacterial pathogen Pseudomonas syringae pv maculicola ES4326. In contrast to cbp60g and sard1 mutations, cbp60a mutations reduced pathogen growth, indicating that CBP60a is a negative regulator of immunity. Bacterial growth was increased by cbp60g only in the presence of CBP60a, while the increase in growth due to sard1 was independent of CBP60a, suggesting that the primary function of CBP60g may be to counter the repressive effect of CBP60a. In the absence of pathogen, levels of SA as well as of several SA-dependent and SA-independent pathogen-inducible genes were higher in cbp60a plants than in the wild type, suggesting that the enhanced resistance of cbp60a plants may result from the activation of immune responses prior to pathogen attack. CBP60a bound calmodulin, and the calmodulin-binding domain was defined at the C-terminal end of the protein. Transgenes encoding mutant versions of CBP60a lacking the ability to bind calmodulin failed to complement null cbp60a mutations, indicating that calmodulin-binding ability is required for the immunity-repressing function of CBP60a. Regulation at the CBP60 node involves negative regulation by CBP60a as well as positive regulation by CBP60g and SARD1, providing multiple levels of control over the activation of immune responses.

Pattern-triggered immunity suppresses programmed cell death triggered by fumonisin b1.

Programmed cell death (PCD) is a crucial process for plant innate immunity and development. In plant innate immunity, PCD is believed to prevent the spread of pathogens from the infection site. Although proper control of PCD is important for plant fitness, we have limited understanding of the molecular mechanisms regulating plant PCD. Plant innate immunity triggered by recognition of effectors (effector-triggered immunity, ETI) is often associated with PCD. However pattern-triggered immunity (PTI), which is triggered by recognition of elicitors called microbe-associated molecular patterns (MAMPs), is not. Therefore we hypothesized that PTI might suppress PCD. Here we report that PCD triggered by the mycotoxin fumonisin B1 (FB1) can be suppressed by PTI in Arabidopsis. FB1-triggered cell death was suppressed by treatment with the MAMPs flg22 (a part of bacterial flagellin) or elf18 (a part of the bacterial elongation factor EF-Tu) but not chitin (a component of fungal cell walls). Although plant hormone signaling is associated with PCD and PTI, both FB1-triggered cell death and suppression of cell death by flg22 treatment were still observed in mutants deficient in jasmonic acid (JA), ethylene (ET) and salicylic acid (SA) signaling. The MAP kinases MPK3 and MPK6 are transiently activated and inactivated within one hour during PTI. We found that FB1 activated MPK3 and MPK6 about 36-48 hours after treatment. Interestingly, this late activation was attenuated by flg22 treatment. These results suggest that PTI suppression of FB1-triggered cell death may involve suppression of MPK3/MPK6 signaling but does not require JA/ET/SA signaling.