Serendipita indica E5'NT modulates extracellular nucleotide levels in the plant apoplast and affects fungal colonization.

Extracellular adenosine 5'-triphosphate (eATP) is an essential signaling molecule that mediates different cellular processes through its interaction with membrane-associated receptor proteins in animals and plants. eATP regulates plant growth, development, and responses to biotic and abiotic stresses. Its accumulation in the apoplast induces ROS production and cytoplasmic calcium increase mediating a defense response to invading microbes. We show here that perception of extracellular nucleotides, such as eATP, is important in plant-fungus interactions and that during colonization by the beneficial root endophyte Serendipita indica eATP accumulates in the apoplast at early symbiotic stages. Using liquid chromatography-tandem mass spectrometry, and cytological and functional analysis, we show that S. indica secrets SiE5'NT, an enzymatically active ecto-5'-nucleotidase capable of hydrolyzing nucleotides in the apoplast. Arabidopsis thaliana lines producing extracellular SiE5'NT are significantly better colonized, have reduced eATP levels, and altered responses to biotic stresses, indicating that SiE5'NT functions as a compatibility factor. Our data suggest that extracellular bioactive nucleotides and their perception play an important role in fungus-root interactions and that fungal-derived enzymes can modify apoplastic metabolites to promote fungal accommodation.

Regulation of related genes promoting resistant in Iris against root rot disease, Fusarium oxysporum f. sp. gladioli.

Iris is one of the most popular and best-selling ornamental plants around the globe. Fusarium root rot disease, Fusarium oxysporum f.sp. gladioli (FOG) is one of the most serious disease of Iridaceae and Iris plants. In this study, three resistant and three susceptible Iris genotypes were inoculated with FOG isolates to evaluate expression of related genes promoting defense to disease at intervals times at two, four and six weeks post inoculation. Total RNA was extracted using an AccuZol™ reagent, and the first-strand Cdna was synthesized accordingly. Expression level of WRKY transcription factors (WRKY), lectin receptor kinase (LecRK), pathogenesis-related protein (PR3), lipoxygenase (LOX1) and ribosome-inactivating proteins (RIP) genes was investigated using quantitative polymerase chain reaction (qPCR). The transcriptional level of five defense-related genes were up-regulated in FOG-infected samples. The genes expression in resistant Iris genotypes NIOP3, NIOP15 and NIOP16 was much higher than susceptible NIOP1, NIOP12 and NIOP20 genotypes. The highest level of expression was observed in all the genes and genotypes at 6 weeks post inoculation. The phenotypic symptoms of genotypes and changes in the expression of genes confirmed resistance in Iris genotypes NIOP3, NIOP15 and NIOP16 in comparison to susceptible genotypes NIOP1, NIOP12 and NIOP20, and un-inoculated control Iris plants. Identifying disease-resistant genotypes can contribute to the development of new ornamental cultivars that can be deployed to ensure high quality and lasting Iris plants.

Ectopic expression of Arabidopsis L-type lectin receptor kinase genes LecRK-I.9 and LecRK-IX.1 in Nicotiana benthamiana confers Phytophthora resistance.

KEY MESSAGE: Transgenic Nicotiana benthamiana lines with constitutive expression of an Arabidopsis lectin receptor kinase gene (LecRK - I.9 or LecRK - IX.1) show enhanced resistance to Phytophthora pathogens, demonstrating conserved gene functionality after interfamily transfer. In plants, cell surface receptors mediate the first layer of innate immunity against pathogenic microbes. In Arabidopsis several L-type lectin receptor kinases (LecRKs) were previously found to function as Phytophthora resistance components. In this study, we determined the functionality of Arabidopsis LecRK-I.9 or LecRK-IX.1 in Phytophthora resistance when transferred into the Solanaceous plant Nicotiana benthamiana. Multiple transgenic lines were generated for each LecRK gene and molecular analyses revealed variation in transgene copy number, transgene expression levels and LecRK protein accumulation. Infection assays showed that transgenic N. benthamiana plants expressing either Arabidopsis LecRK-I.9 or LecRK-IX.1 are more resistant to Phytophthora capsici and to Phytophthora infestans. These results demonstrate that Arabidopsis LecRK-I.9 and LecRK-IX.1 retained their Phytophthora resistance function when transferred into N. benthamiana. Therefore, these LecRKs have the potential to function as a complementary Phytophthora resistance resource in distantly related plant species next to the canonical Phytophthora resistance genes encoding nucleotide-binding leucine-rich repeat proteins.

A novel lectin receptor kinase gene, AtG-LecRK-I.2, enhances bacterial pathogen resistance through regulation of stomatal immunity in Arabidopsis.

The S-locus lectin receptor kinases (G-LecRKs) have been suggested as receptors for microbe/damage-associated molecular patterns (MAMPs/DAMPs) and to be involved in the pathogen defense responses, but the functions of most G-LecRKs in biotic stress response have not been characterized. Here, we identified a member of this family, G-LecRK-I.2, that positively regulates flg22- and Pseudomonas syringae pv. tomato (Pst) DC3000-induced stomatal closure. G-LecRK-I.2 was rapidly phosphorylated under flg22 treatment and could interact with the FLS2/BAK1 complex. Two T-DNA insertion lines, glecrk-i.2-1 and glecrk-i.2-2, had lower levels of reactive oxygen species (ROS) and nitric oxide (NO) production in guard cells, as compared with the wild-type Col-0, under Pst DC3000 infection. Also, the immunity marker genes CBP60g and PR1 were induced at lower levels under Pst DC3000 hrcC- infection in glecrk-i.2-1 and glecrk-i.2-2. The GUS reporter system also revealed that G-LecRK-I.2 was expressed only in guard cells. We also found that G-LecRK-I.2 could interact H+-ATPase AHA1 to regulate H+-ATPase activity in the guard cells. Taken together, our results show that G-LecRK-I.2 plays an important role in regulating stomatal closure under flg22 and Pst DC3000 treatments and in ROS and NO signaling specifically in guard cells.

A Plant Lectin Receptor-like Kinase Phosphorylates the Bacterial Effector AvrPtoB to Dampen Its Virulence in Arabidopsis.

Plasma membrane-localized receptor-like kinases (RLKs) perceive conserved pathogen-associated molecular patterns (PAMPs) in plants, leading to PAMP-triggered immunity (PTI). The Arabidopsis thaliana lectin RLK LecRK-IX.2 has been shown to regulate the bacterial flagellin-derived peptide flg22-induced PTI. Here, we discover that Pseudomonas syringae effector AvrPtoB targets LecRK-IX.2 for degradation, which subsequently suppresses LecRK-IX.2-mediated PTI and disease resistance. However, LecRK-IX.2 can interact with and phosphorylate AvrPtoB at serine site 335 (S335). AvrPtoB self-associates in vitro and in vivo, and the association appears to be essential for its E3 ligase activity in ubiquitinating substrate in plants. Phosphorylation of S335 disrupts the self-association and as a result, phosphomimetic AvrPtoBS335D cannot ubiquitinate LecRK-IX.2 efficiently, leading to the compromised virulence of AvrPtoB in suppressing PTI responses. flg22 enhances AvrPtoB S335 phosphorylation by inducing the expression and activating of LecRK-IX.2. Our study demonstrates that host RLKs can modify pathogen effectors to dampen their virulence and undermine their ability in suppressing PTI.

Extracellular pyridine nucleotides as immune elicitors in arabidopsis.

The pyridine nucleotides nicotinamide adenine dinucleotide (NAD) and NAD phosphate (NADP) are coenzymes that function in both metabolic reactions and intracellular signaling. Emerging evidence from animal research indicates that NAD(P) also acts in the extracellular space (ECS). We have shown in the model plant Arabidopsis that (1) exogenous NAD(P) induces immune responses, (2) pathogen infection causes leakage of intracellular NAD(P) into the extracellular fluid at concentrations sufficient to induce immune responses, and (3) removal of extracellular NAD(P) [eNAD(P)] by expressing the human NAD(P)-metabolizing ectoenzyme CD38 partially compromises systemic acquired resistance. Based on these results, we hypothesize that eNAD(P) is a novel damage-associated molecular pattern (DAMP) in plants; during plant-microbe interaction, intracellular NAD(P) is released from dead or dying cells into the ECS where it interacts with the adjacent healthy cells' surface receptors/targets, which in turn activate downstream specific immune signaling pathways. Our recent identification of LecRK-I.8, a lectin receptor kinase, as the first cell surface NAD+-binding receptor has provided compelling evidence for this hypothesis. Further identification of cell surface eNAD(P) receptors/targets and their downstream signaling components in Arabidopsis as well as determination of the generality of eNAD(P) signaling in crops will help establish eNAD(P) as a conserved DAMP in plants.

At the Frontier; RXLR Effectors Crossing the Phytophthora-Host Interface.

Plants are constantly beset by pathogenic organisms. To successfully infect their hosts, plant pathogens secrete effector proteins, many of which are translocated to the inside of the host cell where they manipulate normal physiological processes and undermine host defense. The way by which effectors cross the frontier to reach the inside of the host cell varies among different classes of pathogens. For oomycete plant pathogens - like the potato late blight pathogen Phytophthora infestans - it has been shown that effector translocation to the host cell cytoplasm is dependent on conserved amino acid motifs that are present in the N-terminal part of effector proteins. One of these motifs, known as the RXLR motif, has a strong resemblance with a host translocation motif found in effectors secreted by Plasmodium species. These malaria parasites, that reside inside specialized vacuoles in red blood cells, make use of a specific protein translocation complex to export effectors from the vacuole into the red blood cell. Whether or not also oomycete RXLR effectors require a translocation complex to cross the frontier is still under investigation. For one P. infestans RXLR effector named IPI-O we have found a potential host target that could play a role in establishing the first contact between this effector and the host cell. This membrane spanning lectin receptor kinase, LecRK-I.9, interacts with IPI-O via the tripeptide RGD that overlaps with the RXLR motif. In animals, RGD is a well-known cell adhesion motif; it binds to integrins, which are membrane receptors that regulate many cellular processes and which can be hijacked by pathogens for either effector translocation or pathogen entry into host cells.