Exploring Pseudomonas syringae pv. tomato biofilm-like aggregate formation in susceptible and PTI-responding Arabidopsis thaliana.

Bacterial biofilm-like aggregates have been observed in plants, but their role in pathogenicity is underinvestigated. In the present study, we observed that extracellular DNA and polysaccharides colocalized with green fluorescent protein (GFP)-expressing Pseudomonas syringae pv. tomato (Pst) aggregates in Arabidopsis leaves, suggesting that Pst aggregates are biofilms. GFP-expressing Pst, Pst ΔalgU ΔmucAB (Pst algU mutant), and Pst ΔalgD ΔalgU ΔmucAB (Pst algU algD mutant) were examined to explore the roles of (1) alginate, a potential biofilm component; (2) Pst AlgU, thought to regulate alginate biosynthesis and some type III secretion system effector genes; and (3) intercellular salicylic acid (SA) accumulation during pathogen-associated molecular pattern-triggered immunity (PTI). Pst formed extensive aggregates in susceptible plants, whereas aggregate numbers and size were reduced in Pst algU and Pst algD algU mutants, and both multiplied poorly in planta, suggesting that aggregate formation contributes to Pst success in planta. However, in SA-deficient sid2-2 plants, Pst algD algU mutant multiplication and aggregate formation were partially restored, suggesting plant-produced SA contributes to suppression of Pst aggregate formation. Pst algD algU mutants formed fewer and smaller aggregates than Pst algU mutants, suggesting both AlgU and AlgD contribute to Pst aggregate formation. Col-0 plants accumulated low levels of SA in response to Pst and both mutants (Pst algU and Pst algD algU), suggesting the regulatory functions of AlgU are not involved in suppressing SA-mediated plant defence. Plant PTI was associated with highly reduced Pst aggregate formation and accumulation of intercellular SA in flg22-induced PTI-responding wild-type Col-0, but not in PTI-incompetent fls2, suggesting intercellular SA accumulation by Arabidopsis contributes to suppression of Pst biofilm-like aggregate formation during PTI.

Metabolite profiling reveals a role for intercellular dihydrocamalexic acid in the response of mature Arabidopsis thaliana to Pseudomonas syringae.

The leaf intercellular space is a site of plant-microbe interactions where pathogenic bacteria such as Pseudomonas syringae grow. In Arabidopsis thaliana, the biosynthesis of tryptophan-derived indolic metabolites is induced by P. syringae infection. Using high-resolution mass spectrometry-based profiling and biosynthetic mutants, we investigated the role of indolic compounds and other small molecules in the response of mature Arabidopsis to P. syringae. We observed dihydrocamalexic acid (DHCA), the precursor to the defense-related compound camalexin, accumulating in intercellular washing fluids (IWFs) without further conversion to camalexin. The indolic biosynthesis mutant cyp71a12/cyp71a13 was more susceptible to P. syringae compared to mature wild-type plants displaying age-related resistance (ARR). DHCA and structural analogs inhibit P. syringae growth (MIC ~ 500 µg/mL), but not at concentrations found in IWFs, and DHCA did not inhibit biofilm formation in vitro. However, infiltration of exogenous DHCA enhanced resistance in mature cyp71a12/cyp71a13. These results provide evidence that DHCA derived from CYP71A12 and CYP71A13 activity accumulates in the intercellular space and contributes to the resistance of mature Arabidopsis to P. syringae without directly inhibiting bacterial growth.

Systemic acquired resistance networks amplify airborne defense cues.

Salicylic acid (SA)-mediated innate immune responses are activated in plants perceiving volatile monoterpenes. Here, we show that monoterpene-associated responses are propagated in feed-forward loops involving the systemic acquired resistance (SAR) signaling components pipecolic acid, glycerol-3-phosphate, and LEGUME LECTIN-LIKE PROTEIN1 (LLP1). In this cascade, LLP1 forms a key regulatory unit in both within-plant and between-plant propagation of immunity. The data integrate molecular components of SAR into systemic signaling networks that are separate from conventional, SA-associated innate immune mechanisms. These networks are central to plant-to-plant propagation of immunity, potentially raising SAR to the population level. In this process, monoterpenes act as microbe-inducible plant volatiles, which as part of plant-derived volatile blends have the potential to promote the generation of a wave of innate immune signaling within canopies or plant stands. Hence, plant-to-plant propagation of SAR holds significant potential to fortify future durable crop protection strategies following a single volatile trigger.

Corrigendum: Orthology Analysis and In Vivo Complementation Studies to Elucidate the Role of DIR1 during Systemic Acquired Resistance in Arabidopsis thaliana and Cucumis sativus.

[This corrects the article on p. 566 in vol. 7, PMID: 27200039.].

Age-Related Resistance in Arabidopsis thaliana Involves the MADS-Domain Transcription Factor SHORT VEGETATIVE PHASE and Direct Action of Salicylic Acid on Pseudomonas syringae.

Arabidopsis thaliana exhibits a developmentally regulated disease-resistance response known as age-related resistance (ARR), a process that requires intercellular accumulation of salicylic acid (SA), which is thought to act as an antimicrobial agent. ARR is characterized by enhanced resistance to some pathogens at the late adult-vegetative and reproductive stages. While the transition to flowering does not cause the onset of ARR, both processes involve the MADS-domain transcription factor SHORT VEGETATIVE PHASE (SVP). In this study, ARR-defective svp mutants were found to accumulate reduced levels of intercellular SA compared with wild type in response to Pseudomonas syringae pv. tomato. Double mutant and overexpression analyses suggest that SVP and SOC1 (SUPPRESSOR OF OVEREXPRESSION OF CO 1) act antagonistically, such that SVP is required for ARR to alleviate the negative effects of SOC1 on SA accumulation. In vitro, SA exhibited antibacterial and antibiofilm activity at concentrations similar to those measured in the intercellular space during ARR. In vivo, P. syringae pv. tomato formed biofilm-like aggregates in young susceptible plants, while this was drastically reduced in mature ARR-competent plants, which accumulate intercellular SA. Collectively, these results reveal a novel role for the floral regulators SVP and SOC1 in disease resistance and provide evidence that SA acts directly on pathogens as an antimicrobial agent. [Formula: see text] Copyright © 2017 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Vascular Sap Proteomics: Providing Insight into Long-Distance Signaling during Stress.

The plant vascular system, composed of the xylem and phloem, is important for the transport of water, mineral nutrients, and photosynthate throughout the plant body. The vasculature is also the primary means by which developmental and stress signals move from one organ to another. Due to practical and technological limitations, proteomics analysis of xylem and phloem sap has been understudied in comparison to accessible sample types such as leaves and roots. However, recent advances in sample collection techniques and mass spectrometry technology are making it possible to comprehensively analyze vascular sap proteomes. In this mini-review, we discuss the emerging field of vascular sap proteomics, with a focus on recent comparative studies to identify vascular proteins that may play roles in long-distance signaling and other processes during stress responses in plants.

Comparative Proteomics Analysis of Phloem Exudates Collected during the Induction of Systemic Acquired Resistance.

Systemic acquired resistance (SAR) is a plant defense response that provides long-lasting, broad-spectrum pathogen resistance to uninfected systemic leaves following an initial localized infection. In Arabidopsis (Arabidopsis thaliana), local infection with virulent or avirulent strains of Pseudomonas syringae pv tomato generates long-distance SAR signals that travel from locally infected to distant leaves through the phloem to establish SAR In this study, a proteomics approach was used to identify proteins that accumulate in phloem exudates in response to the induction of SAR To accomplish this, phloem exudates collected from mock-inoculated or SAR-induced leaves of wild-type Columbia-0 plants were subjected to label-free quantitative liquid chromatography-tandem mass spectrometry proteomics. Comparing mock- and SAR-induced phloem exudate proteomes, 16 proteins were enriched in phloem exudates collected from SAR-induced plants, while 46 proteins were suppressed. SAR-related proteins THIOREDOXIN h3, ACYL-COENZYME A-BINDING PROTEIN6, and PATHOGENESIS-RELATED1 were enriched in phloem exudates of SAR-induced plants, demonstrating the strength of this approach and suggesting a role for these proteins in the phloem during SAR To identify novel components of SAR, transfer DNA mutants of differentially abundant phloem proteins were assayed for SAR competence. This analysis identified a number of new proteins (m-type thioredoxins, major latex protein-like protein, ULTRAVIOLET-B RESISTANCE8 photoreceptor) that contribute to the SAR response. The Arabidopsis SAR phloem proteome is a valuable resource for understanding SAR long-distance signaling and the dynamic nature of the phloem during plant-pathogen interactions.

Orthology Analysis and In Vivo Complementation Studies to Elucidate the Role of DIR1 during Systemic Acquired Resistance in Arabidopsis thaliana and Cucumis sativus.

AtDIR1 (Defective in Induced Resistance1) is an acidic lipid transfer protein essential for systemic acquired resistance (SAR) in Arabidopsis thaliana. Upon SAR induction, DIR1 moves from locally infected to distant uninfected leaves to activate defense priming; however, a molecular function for DIR1 has not been elucidated. Bioinformatic analysis and in silico homology modeling identified putative AtDIR1 orthologs in crop species, revealing conserved protein motifs within and outside of DIR1's central hydrophobic cavity. In vitro assays to compare the capacity of recombinant AtDIR1 and targeted AtDIR1-variant proteins to bind the lipophilic probe TNS (6,P-toluidinylnaphthalene-2-sulfonate) provided evidence that conserved leucine 43 and aspartic acid 39 contribute to the size of the DIR1 hydrophobic cavity and possibly hydrophobic ligand binding. An Arabidopsis-cucumber SAR model was developed to investigate the conservation of DIR1 function in cucumber (Cucumis sativus), and we demonstrated that phloem exudates from SAR-induced cucumber rescued the SAR defect in the Arabidopsis dir1-1 mutant. Additionally, an AtDIR1 antibody detected a protein of the same size as AtDIR1 in SAR-induced cucumber phloem exudates, providing evidence that DIR1 function during SAR is conserved in Arabidopsis and cucumber. In vitro TNS displacement assays demonstrated that recombinant AtDIR1 did not bind the SAR signals azelaic acid (AzA), glycerol-3-phosphate or pipecolic acid. However, recombinant CsDIR1 and CsDIR2 interacted weakly with AzA and pipecolic acid. Bioinformatic and functional analyses using the Arabidopsis-cucumber SAR model provide evidence that DIR1 orthologs exist in tobacco, tomato, cucumber, and soybean, and that DIR1-mediated SAR signaling is conserved in Arabidopsis and cucumber.

Mind the gap: Signal movement through plasmodesmata is critical for the manifestation of SAR.

Systemic acquired resistance (SAR) is a plant defense response in which an initial localized infection affords enhanced pathogen resistance to distant, uninfected leaves. SAR requires efficient long-distance signaling between the infected leaf, where SAR signals are generated, and the distant uninfected leaves that receive them. A growing body of evidence indicates that the lipid transfer protein DIR1 (Defective in Induced Resistance) is an important mediator of long-distance SAR signaling. In a recent publication, we investigated if cell-to-cell movement through plasmodesmata is required for long-distance movement of DIR1 during SAR. We determined that overexpression of Plasmodesmata-Located Proteins (PDLP1 and 5) negatively impacted long-distance DIR1 movement and SAR competence, suggesting that movement through plasmodesmata contributes to long-distance signal movement during SAR.

Investigation of intercellular salicylic acid accumulation during compatible and incompatible Arabidopsis-pseudomonas syringae interactions using a fast neutron-generated mutant allele of EDS5 identified by genetic mapping and whole-genome sequencing.

A whole-genome sequencing technique developed to identify fast neutron-induced deletion mutations revealed that iap1-1 is a new allele of EDS5 (eds5-5). RPS2-AvrRpt2-initiated effector-triggered immunity (ETI) was compromised in iap1-1/eds5-5 with respect to in planta bacterial levels and the hypersensitive response, while intra- and intercellular free salicylic acid (SA) accumulation was greatly reduced, suggesting that SA contributes as both an intracellular signaling molecule and an antimicrobial agent in the intercellular space during ETI. During the compatible interaction between wild-type Col-0 and virulent Pseudomonas syringae pv. tomato (Pst), little intercellular free SA accumulated, which led to the hypothesis that Pst suppresses intercellular SA accumulation. When Col-0 was inoculated with a coronatine-deficient strain of Pst, high levels of intercellular SA accumulation were observed, suggesting that Pst suppresses intercellular SA accumulation using its phytotoxin coronatine. This work suggests that accumulation of SA in the intercellular space is an important component of basal/PAMP-triggered immunity as well as ETI to pathogens that colonize the intercellular space.

Some things get better with age: differences in salicylic acid accumulation and defense signaling in young and mature Arabidopsis.

In Arabidopsis, much of what we know about the phytohormone salicylic acid (SA) and its role in plant defense comes from experiments using young plants. We are interested in understanding why young plants are susceptible to virulent strains of Pseudomonas syringae, while mature plants exhibit a robust defense response known as age-related resistance (ARR). SA-mediated signaling is important for defense in young plants, however, ARR occurs independently of the defense regulators NPR1 and WHY1. Furthermore, intercellular SA accumulation is an important component of ARR, and intercellular washing fluids from ARR-competent plants exhibit antibacterial activity, suggesting that SA acts as an antimicrobial agent in the intercellular space. Young plants accumulate both intracellular and intercellular SA during PAMP- and effector-triggered immunity, however, virulent P. syringae promotes susceptibility by suppressing SA accumulation using the phytotoxin coronatine. Here we outline the hypothesis that mature, ARR-competent Arabidopsis alleviates coronatine-mediated suppression of SA accumulation. We also explore the role of SA in other mature-plant processes such as flowering and senescence, and discuss their potential impact on ARR.

Intercellular salicylic acid accumulation during compatible and incompatible Arabidopsis-Pseudomonas syringae interactions.

The phytohormone salicylic acid (SA) plays an important role in several disease resistance responses. During the Age-Related Resistance (ARR) response that occurs in mature Arabidopsis responding to Pseudomonas syringae pv tomato (Pst), SA accumulates in the intercellular space where it may act as an antimicrobial agent. Recently we measured intracellular and intercellular SA levels in young, ARR-incompetent plants responding to virulent and avirulent strains of Pst to determine if intercellular SA accumulation is a component of additional defense responses to Pst. In young plants virulent Pst suppressed both intra- and intercellular SA accumulation in a coronatine-dependent manner. In contrast, high levels of intra- and intercellular SA accumulated in response to avirulent Pst. Our results support the idea that SA accumulation in the intercellular space is an important component of multiple defense responses. Future research will include understanding how mature plants counteract the effects of coronatine during the ARR response.

RNA-Seq effectively monitors gene expression in Eutrema salsugineum plants growing in an extreme natural habitat and in controlled growth cabinet conditions.

BACKGROUND: The investigation of extremophile plant species growing in their natural environment offers certain advantages, chiefly that plants adapted to severe habitats have a repertoire of stress tolerance genes that are regulated to maximize plant performance under physiologically challenging conditions. Accordingly, transcriptome sequencing offers a powerful approach to address questions concerning the influence of natural habitat on the physiology of an organism. We used RNA sequencing of Eutrema salsugineum, an extremophile relative of Arabidopsis thaliana, to investigate the extent to which genetic variation and controlled versus natural environments contribute to differences between transcript profiles. RESULTS: Using 10 million cDNA reads, we compared transcriptomes from two natural Eutrema accessions (originating from Yukon Territory, Canada and Shandong Province, China) grown under controlled conditions in cabinets and those from Yukon plants collected at a Yukon field site. We assessed the genetic heterogeneity between individuals using single-nucleotide polymorphisms (SNPs) and the expression patterns of 27,016 genes. Over 39,000 SNPs distinguish the Yukon from the Shandong accessions but only 4,475 SNPs differentiated transcriptomes of Yukon field plants from an inbred Yukon line. We found 2,989 genes that were differentially expressed between the three sample groups and multivariate statistical analyses showed that transcriptomes of individual plants from a Yukon field site were as reproducible as those from inbred plants grown under controlled conditions. Predicted functions based upon gene ontology classifications show that the transcriptomes of field plants were enriched by the differential expression of light- and stress-related genes, an observation consistent with the habitat where the plants were found. CONCLUSION: Our expectation that comparative RNA-Seq analysis of transcriptomes from plants originating in natural habitats would be confounded by uncontrolled genetic and environmental factors was not borne out. Moreover, the transcriptome data shows little genetic variation between laboratory Yukon Eutrema plants and those found at a field site. Transcriptomes were reproducible and biological associations meaningful whether plants were grown in cabinets or found in the field. Thus RNA-Seq is a valuable approach to study native plants in natural environments and this technology can be exploited to discover new gene targets for improved crop performance under adverse conditions.

Long distance movement of DIR1 and investigation of the role of DIR1-like during systemic acquired resistance in Arabidopsis.

DIR1 is a lipid transfer protein (LTP) postulated to complex with and/or chaperone a signal(s) to distant leaves during Systemic Acquired Resistance (SAR) in Arabidopsis. DIR1 was detected in phloem sap-enriched petiole exudates collected from wild-type leaves induced for SAR, suggesting that DIR1 gains access to the phloem for movement from the induced leaf. Occasionally the defective in induced resistance1 (dir1-1) mutant displayed a partially SAR-competent phenotype and a DIR1-sized band in protein gel blots was detected in dir1-1 exudates suggesting that a highly similar protein, DIR1-like (At5g48490), may contribute to SAR. Recombinant protein studies demonstrated that DIR1 polyclonal antibodies recognize DIR1 and DIR1-like. Homology modeling of DIR1-like using the DIR1-phospholipid crystal structure as template, provides clues as to why the dir1-1 mutant is rarely SAR-competent. The contribution of DIR1 and DIR1-like during SAR was examined using an Agrobacterium-mediated transient expression-SAR assay and an estrogen-inducible DIR1-EGFP/dir1-1 line. We provide evidence that upon SAR induction, DIR1 moves down the leaf petiole to distant leaves. Our data also suggests that DIR1-like displays a reduced capacity to move to distant leaves during SAR and this may explain why dir1-1 is occasionally SAR-competent.

The floral transition is not the developmental switch that confers competence for the Arabidopsis age-related resistance response to Pseudomonas syringae pv. tomato.

Age-related resistance (ARR) is a plant defense response characterized by enhanced resistance to certain pathogens in mature plants relative to young plants. In Arabidopsis thaliana the transition to flowering is associated with ARR competence, suggesting that this developmental event is the switch that initiates ARR competence in mature plants (Rusterucci et al. in Physiol Mol Plant Pathol 66:222-231, 2005). The association of ARR and the floral transition was examined using flowering-time mutants and photoperiod-induced flowering to separate flowering from other developmental events that occur as plants age. Under short-day conditions, late-flowering plant lines ld-1 (luminidependens-1), soc1-2 (suppressor of overexpression of co 1-2), and FRI (+) (FRIGIDA) displayed ARR before the transition to flowering occurred. Early-flowering svp-31, svp-32 (short vegetative phase), and Ws-2 were ARR-defective, whereas early-flowering tfl1-14 (terminal flower 1-14) displayed ARR at the same time as Col-0. While svp-31, svp-32 and Ws-2 produced few rosette leaves, tfl1-14 produced a rosette leaf number similar to Col-0, suggesting that the development of a minimum number of rosette leaves is necessary to initiate ARR competence under short-day conditions. Photoperiod-induced transient expression of FT (FLOWERING LOCUS T) caused precocious flowering in short-day-grown Col-0 but this was not associated with ARR competence. Under long-day conditions co-9 (constans-9) mutants did not flower but displayed an ARR response at the same time as Col-0. This study suggests that SVP is required for the ARR response and that the floral transition is not the developmental event that regulates ARR competence.

Localization of DIR1 at the tissue, cellular and subcellular levels during Systemic Acquired Resistance in Arabidopsis using DIR1:GUS and DIR1:EGFP reporters.

BACKGROUND: Systemic Acquired Resistance (SAR) is an induced resistance response to pathogens, characterized by the translocation of a long-distance signal from induced leaves to distant tissues to prime them for increased resistance to future infection. DEFECTIVE in INDUCED RESISTANCE 1 (DIR1) has been hypothesized to chaperone a small signaling molecule to distant tissues during SAR in Arabidopsis. RESULTS: DIR1 promoter:DIR1-GUS/dir1-1 lines were constructed to examine DIR1 expression. DIR1 is expressed in seedlings, flowers and ubiquitously in untreated or mock-inoculated mature leaf cells, including phloem sieve elements and companion cells. Inoculation of leaves with SAR-inducing avirulent or virulent Pseudomonas syringae pv tomato (Pst) resulted in Type III Secretion System-dependent suppression of DIR1 expression in leaf cells. Transient expression of fluorescent fusion proteins in tobacco and intercellular washing fluid experiments indicated that DIR1's ER signal sequence targets it for secretion to the cell wall. However, DIR1 expressed without a signal sequence rescued the dir1-1 SAR defect, suggesting that a cytosolic pool of DIR1 is important for the SAR response. CONCLUSIONS: Although expression of DIR1 decreases during SAR induction, the protein localizes to all living cell types of the vasculature, including companion cells and sieve elements, and therefore DIR1 is well situated to participate in long-distance signaling during SAR.

Forward and reverse genetics to identify genes involved in the age-related resistance response in Arabidopsis thaliana.

SUMMARY Age-related resistance (ARR) occurs in numerous plant species, often resulting in increased disease resistance as plants mature. ARR in Arabidopsis to Pseudomonas syringae pv. tomato is associated with intercellular salicylic acid (SA) accumulation and the transition to flowering. Forward and reverse genetic screens were performed to identify genes required for ARR and to investigate the mechanism of the ARR response. Infiltration of SA into the intercellular space of the ARR-defective mutant iap1-1 (important for the ARR pathway) partially restored ARR function. Inter- and intracellular SA accumulation was reduced in the mutant iap1-1 compared with the wild-type, and the SA regulatory gene EDS1 was also required for ARR. Combining microarray analysis with reverse genetics using T-DNA insertion lines, four additional ARR genes were identified as contributing to ARR: two plant-specific transcription factors of the NAC family [ANAC055 (At3g15500) and ANAC092 (At5g39610)], a UDP-glucose glucosyltransferase [UGT85A1 (At1g22400)] and a cytidine deaminase [CDA1 (At2g19570)]. These four genes and IAP1 are also required for ARR to Hyaloperonospora parasitica. IAP1 encodes a key component of ARR that acts upstream of SA accumulation and possibly downstream of UGT85A1, CDA1 and the two NAC transcription factors (ANAC055, ANAC092).

Identification of likely orthologs of tobacco salicylic acid-binding protein 2 and their role in systemic acquired resistance in Arabidopsis thaliana.

Salicylic acid-binding protein 2 (SABP2) is essential for the establishment of systemic acquired resistance (SAR) in tobacco; SABP2's methyl salicylate (MeSA) esterase activity is required in healthy systemic tissues of infected plants to release the active defense phytohormone SA from MeSA, which serves as a long-distance signal for SAR. In the current study, we characterize a new gene family from Arabidopsis thaliana encoding 18 potentially active alpha/beta fold hydrolases that share 32-57% identity with SABP2. Of 14 recombinant AtMES (MES for methyl esterase) proteins tested, five showed preference for MeSA as a substrate and displayed SA inhibition of MeSA esterase activity in vitro (AtMES1, -2, -4, -7, and -9). The two genes encoding MeSA esterases with the greatest activity, AtMES1 and -9, as well as AtMES7 were transcriptionally upregulated during infection of Arabidopsis with avirulent Pseudomonas syringae. In addition, conditional expression of AtMES1, -7, or -9 complemented SAR deficiency in SABP2-silenced tobacco, suggesting that these three members of the AtMES family are SABP2 functional homologs (orthologs). Underexpression by knockout mutation and/or RNAi-mediated silencing of multiple AtMES genes, including AtMES1, -2, -7, and -9, compromised SAR in Arabidopsis and correlated with enhanced accumulation of MeSA in the systemic tissue of SAR-induced plants. Together, the data show that several members of the AtMES gene family are functionally homologous to SABP2 and redundant for MeSA hydrolysis and probably SAR. These data suggest that MeSA is a conserved SAR signal in Arabidopsis and tobacco.

Altered Expression of PERK Receptor Kinases in Arabidopsis Leads to Changes in Growth and Floral Organ Formation.

The proline-rich, extensin-like receptor kinase (PERK) family is characterized by a putative extracellular domain related to cell wall proteins, followed by a transmembrane domain and kinase domain. The original member, PERK1, was isolated from Brassica napus (BnPERK1) and 15 PERK1-related members were subsequently identified in the Arabidopsis thaliana genome. Ectopic expression and antisense suppression studies were performed using the BnPERK1 cDNA under the control of the 35S CaMV constitutive promoter and introduced into Arabidopsis. In the case of antisense suppression, the BnPERK1 cDNA shared sufficient sequence similarity to suppress several members of the At PERK family. In both sets of transgenic Arabidopsis, several heritable changes in growth and development were observed. Antisense BnPERK1 transgenic Arabidopsis showed various growth defects including loss of apical dominance, increased secondary branching, and floral organ defects. In contrast, Arabidopsis plants ectopically expressing BnPERK1 displayed a prolonged lifespan with increased lateral shoot production and seed set. Along with these phenotypic changes, aberrant deposits of callose and cellulose were also observed, suggestive of cell wall changes as a consequence of altered PERK expression.

A comprehensive expression analysis of the Arabidopsis proline-rich extensin-like receptor kinase gene family using bioinformatic and experimental approaches.

The Arabidopsis proline-rich extensin-like receptor kinase (PERK) family consists of 15 predicted receptor kinases. A comprehensive expression analysis was undertaken to identify overlapping and unique expression patterns within this family relative to their phylogeny. Three different approaches were used to study AtPERK gene family expression, and included analyses of the EST, MPSS and NASCArrays databases as well as experimental RNA blot analyses. Some of the AtPERK members were identified as tissue-specific genes while others were more broadly expressed. While in some cases there was a good association between these different expression patterns and the position of the AtPERK members in the kinase phylogeny, in other cases divergence of expression patterns was seen. The PERK expression data identified by the bioinformatics and experimental approaches were found generally to show similar trends and supported the use of data from large-scale expression studies for obtaining preliminary expression data. Thus, the bioinformatics survey for ESTs and microarrays is a powerful comprehensive approach for obtaining a genome-wide view of genes in a multigene family.