The Arabidopsis Wall Associated Kinase-Like 10 Gene Encodes a Functional Guanylyl Cyclase and Is Co-Expressed with Pathogen Defense Related Genes

Background

Second messengers have a key role in linking environmental stimuli to physiological responses. One such messenger, guanosine 3′,5′-cyclic monophosphate (cGMP), has long been known to be an essential signaling molecule in many different physiological processes in higher plants, including biotic stress responses. To date, however, the guanylyl cyclase (GC) enzymes that catalyze the formation of cGMP from GTP have largely remained elusive in higher plants.

Principal Findings

We have identified an Arabidopsis receptor type wall associated kinase–like molecule (AtWAKL10) as a candidate GC and provide experimental evidence to show that the intracellular domain of AtWAKL10_431–700 can generate cGMP in vitro. Further, we also demonstrate that the molecule has kinase activity indicating that AtWAKL10 is a twin-domain catalytic protein. A co-expression and stimulus-specific expression analysis revealed that AtWAKL10 is consistently co-expressed with well characterized pathogen defense related genes and along with these genes is induced early and sharply in response to a range of pathogens and their elicitors.

Conclusions

We demonstrate that AtWAKL10 is a twin-domain, kinase-GC signaling molecule that may function in biotic stress responses that are critically dependent on the second messenger cGMP.     

Ginkgo biloba responds to herbivory by activating early signaling and direct defenses

Background

Ginkgo biloba (Ginkgoaceae) is one of the most ancient living seed plants and is regarded as a living fossil. G. biloba has a broad spectrum of resistance or tolerance to many pathogens and herbivores because of the presence of toxic leaf compounds. Little is known about early and late events occurring in G. biloba upon herbivory. The aim of this study was to assess whether herbivory by the generalist Spodoptera littoralis was able to induce early signaling and direct defense in G. biloba by evaluating early and late responses.

Methodology/Principal Findings

Early and late responses in mechanically wounded leaves and in leaves damaged by S. littoralis included plasma transmembrane potential (Vm) variations, time-course changes in both cytosolic calcium concentration ([Ca²⁺]_cyt) and H₂O₂ production, the regulation of genes correlated to terpenoid and flavonoid biosynthesis, the induction of direct defense compounds, and the release of volatile organic compounds (VOCs). The results show that G. biloba responded to hebivory with a significant Vm depolarization which was associated to significant increases in both [Ca²⁺]_cyt and H₂O₂. Several defense genes were regulated by herbivory, including those coding for ROS scavenging enzymes and the synthesis of terpenoids and flavonoids. Metabolomic analyses revealed the herbivore-induced production of several flavonoids and VOCs. Surprisingly, no significant induction by herbivory was found for two of the most characteristic G. biloba classes of bioactive compounds; ginkgolides and bilobalides.

Conclusions/Significance

By studying early and late responses of G. biloba to herbivory, we provided the first evidence that this “living fossil” plant responds to herbivory with the same defense mechanisms adopted by the most recent angiosperms.     

Raphanusanin-mediated resistance to pathogens is light dependent in radish and Arabidopsis thaliana

Raphanusanin (Ra) is a light-induced inhibitor of hypocotyl growth that responds to unilateral blue light illumination in radish seedlings. We have previously shown that Ra regulates genes that are involved in common defense mechanisms. Many genes that are induced by Ra are also positively regulated by early blue light. To extend the understanding of the role of Ra in pathogen defense, we evaluated the effects of Ra on radish and Arabidopsis thaliana (A. thaliana) infected with the necrotrophic pathogen Botrytis cinerea (B. cinerea) and biotrophic pathogen Pseudomonas syringae (P. syringae). Radish and A. thaliana were found to be resistant to both pathogens when treated with Ra, depending on the concentration used. Interestingly, Ra-mediated resistance to P. syringae is dependent on light because Ra-treated seedlings exhibited enhanced susceptibility to P. syringae infection when grown in the dark. In addition to regulating the biotic defense response, Ra inhibited seed germination and root elongation and enhanced the growth of root hairs in the presence of light in radish and A. thaliana. Our data suggest that Ra regulates the expression of a set of genes involved in defense signaling pathways and plays a role in pathogen defense and plant development. Our results show that light may be generally required not only for the accumulation of Ra but also for its activation during the pathogen defense response.     

Comparison of antibody repertoires produced by HIV-1 infection, other chronic and acute infections, and systemic autoimmune disease

Background

Antibodies (Abs) produced during HIV-1 infection rarely neutralize a broad range of viral isolates; only eight broadly-neutralizing (bNt) monoclonal (M)Abs have been isolated. Yet, to be effective, an HIV-1 vaccine may have to elicit the essential features of these MAbs. The V genes of all of these bNt MAbs are highly somatically mutated, and the V_H genes of five of them encode a long (≥20 aa) third complementarity-determining region (CDR-H3). This led us to question whether long CDR-H3s and high levels of somatic mutation (SM) are a preferred feature of anti-HIV bNt MAbs, or if other adaptive immune responses elicit them in general.

Methodology and Principal Findings

We assembled a V_H-gene sequence database from over 700 human MAbs of known antigen specificity isolated from chronic (viral) infections (ChI), acute (bacterial and viral) infections (AcI), and systemic autoimmune diseases (SAD), and compared their CDR-H3 length, number of SMs and germline V_H-gene usage. We found that anti-HIV Abs, regardless of their neutralization breadth, tended to have long CDR-H3s and high numbers of SMs. However, these features were also common among Abs associated with other chronic viral infections. In contrast, Abs from acute viral infections (but not bacterial infections) tended to have relatively short CDR-H3s and a low number of SMs, whereas SAD Abs were generally intermediate in CDR-H3 length and number of SMs. Analysis of V_H gene usage showed that ChI Abs also tended to favor distal germline V_H-genes (particularly V_H1-69), especially in Abs bearing long CDR-H3s.

Conclusions and Significance

The striking difference between the Abs produced during chronic vs. acute viral infection suggests that Abs bearing long CDR-H3s, high levels of SM and V_H1-69 gene usage may be preferentially selected during persistent infection.     

Characterization of Multisugar-Binding C-Type Lectin (SpliLec) from a Bacterial-Challenged Cotton Leafworm, Spodoptera littoralis

Background

Various proteins that display carbohydrate-binding activity in a Ca²⁺-dependent manner are classified into the C-type lectin family. They have one or two C-type carbohydrate-recognition domains (CRDs) composed of 110–130 amino acid residues in common. C-type lectins mediate cell adhesion, non-self recognition, and immuno-protection processes in immune responses and thus play significant roles in clearance of invaders, either as cell surface receptors for microbial carbohydrates or as soluble proteins existing in tissue fluids. The lectin of Spodoptera littoralis is still uncharacterized.

Methodology

A single orf encoding a deduced polypeptide consisting of an 18-residue signal peptide and a 291-residue mature peptide, termed SpliLec, was isolated from the haemolymph of the cotton leafworm, S. littoralis, after bacterial challenge using RACE-PCR. Sequence analyses of the data revealed that SpliLec consists of two CRDs. Short-form CRD₁ and long-form CRD₂ are stabilized by two and three highly conserved disulfide bonds, respectively. SpliLec shares homology with some dipteran lectins suggesting possible common ancestor. The purified SpliLec exhibited a 140-kDa molecular mass with a subunit molecular mass of 35 kDa. The hemagglutination assays of the SpliLec confirmed a thermally stable, multisugar-binding C-type lectin that binds different erythrocytes. The purified SpliLec agglutinated microorganisms and exhibited comparable antimicrobial activity against gram (+) and gram (−) bacteria too.

Conclusions

Our results suggested an important role of the SpliLec gene in cell adhesion and non-self recognition. It may cooperate with other AMPs in clearance of invaders of Spodoptera littoralis.     

Silencing of aphid genes by dsRNA feeding from plants

Background

RNA interference (RNAi) is a valuable reverse genetics tool to study gene function in various organisms, including hemipteran insects such as aphids. Previous work has shown that RNAi-mediated knockdown of pea aphid (Acyrthosiphon pisum) genes can be achieved through direct injection of double-stranded RNA (dsRNA) or small-interfering RNAs (siRNA) into the pea aphid hemolymph or by feeding these insects on artificial diets containing the small RNAs.

Methodology/Principal Findings

In this study, we have developed the plant-mediated RNAi technology for aphids to allow for gene silencing in the aphid natural environment and minimize handling of these insects during experiments. The green peach aphid M. persicae was selected because it has a broad plant host range that includes the model plants Nicotiana benthamiana and Arabidopsis thaliana for which transgenic materials can relatively quickly be generated. We targeted M. persicae Rack1, which is predominantly expressed in the gut, and M. persicae C002 (MpC002), which is predominantly expressed in the salivary glands. The aphids were fed on N. benthamiana leaf disks transiently producing dsRNA corresponding to these genes and on A. thaliana plants stably producing the dsRNAs. MpC002 and Rack-1 expression were knocked down by up to 60% on transgenic N. benthamiana and A. thaliana. Moreover, silenced M. persicae produced less progeny consistent with these genes having essential functions.

Conclusions/Significance

Similar levels of gene silencing were achieved in our plant-mediated RNAi approach and published silencing methods for aphids. Furthermore, the N. benthamiana leaf disk assay can be developed into a screen to assess which genes are essential for aphid survival on plants. Our results also demonstrate the feasibility of the plant-mediated RNAi approach for aphid control.     

Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato

Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in this process are currently unclear. Here, we report that Arabidopsis thaliana UGT76B1 generated glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants did not accumulate NHP-Glc and accumulated less glycosylated salicylic acid (SA-Glc) than wild-type plants. The metabolic changes in ugt76b1 plants were accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of the SAR molecules NHP and salicylic acid by UGT76B1 plays an important role in modulating defense responses. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato (Solanum lycopersicum) increased NHP-Glc production and reduced NHP accumulation in local tissue and abolished the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.

The Arabidopsis UDP-glycosyltransferase UGT76B1 glycosylates the systemic acquired resistance-signaling metabolite NHP and can inactivate systemic defense responses when expressed in tomato.     

Agroinfiltration Reduces ABA Levels and Suppresses Pseudomonas syringae-Elicited Salicylic Acid Production in Nicotiana tabacum

Background

Agrobacterium tumefaciens strain GV3101 (pMP90) is widely used in transient gene expression assays, including assays to study pathogen effectors and plant disease resistance mechanisms. However, inoculation of A. tumefaciens GV3101 into Nicotiana tabacum (tobacco) leaves prior to infiltration with pathogenic and non-host strains of Pseudomonas syringae results in suppression of macroscopic symptoms when compared with leaves pre-treated with a buffer control.

Methodology/Findings

To gain further insight into the mechanistic basis of symptom suppression by A. tumefaciens we examined the effect of pre-treatment with A. tumefaciens on the growth of P. syringae, the production of the plant signalling molecules salicylic acid (SA) and abscisic acid (ABA), and the presence of callose deposits. Pre-treatment with A. tumefaciens reduced ABA levels, P. syringae multiplication and P. syringae-elicited SA and ABA production, but promoted increased callose deposition. However, pre-treatment with A. tumefaciens did not suppress necrosis or SA production in leaves inoculated with the elicitor HrpZ.

Conclusions/Significance

Collectively, these results show that inoculation of N. tabacum leaves with A. tumefaciens alters plant hormone levels and plant defence responses to P. syringae, and demonstrate that researchers should consider the impact of A. tumefaciens on plant signal transduction when using A. tumefaciens-mediated transient expression assays to investigate ABA-regulated processes or pathogenicity and plant defence mechanisms.     

Transcriptomic analysis reveals tomato genes whose expression is induced specifically during effector-triggered immunity and identifies the Epk1 protein kinase which is required for the host response to three bacterial effector proteins

Background

Plants have two related immune systems to defend themselves against pathogen attack. Initially, pattern-triggered immunity is activated upon recognition of microbe-associated molecular patterns by pattern recognition receptors. Pathogenic bacteria deliver effector proteins into the plant cell that interfere with this immune response and promote disease. However, some plants express resistance proteins that detect the presence of specific effectors leading to a robust defense response referred to as effector-triggered immunity. The interaction of tomato with Pseudomonas syringae pv. tomato is an established model system for understanding the molecular basis of these plant immune responses.

Results

We apply high-throughput RNA sequencing to this pathosystem to identify genes whose expression changes specifically during pattern-triggered or effector-triggered immunity. We then develop reporter genes for each of these responses that will enable characterization of the host response to the large collection of P. s. pv. tomato strains that express different combinations of effectors. Virus-induced gene silencing of 30 of the effector-triggered immunity-specific genes identifies Epk1 which encodes a predicted protein kinase from a family previously unknown to be involved in immunity. Knocked-down expression of Epk1 compromises effector-triggered immunity triggered by three bacterial effectors but not by effectors from non-bacterial pathogens. Epistasis experiments indicate that Epk1 acts upstream of effector-triggered immunity-associated MAP kinase signaling.

Conclusions

Using RNA-seq technology we identify genes involved in specific immune responses. A functional genomics screen led to the discovery of Epk1, a novel predicted protein kinase required for plant defense activation upon recognition of three different bacterial effectors.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-014-0492-1) contains supplementary material, which is available to authorized users.