The Polyamine Putrescine Contributes to H2O2 and RbohD/F-Dependent Positive Feedback Loop in Arabidopsis PAMP-Triggered Immunity

Polyamines are involved in defense against pathogenic microorganisms in plants. However, the role of the polyamine putrescine (Put) during plant defense has remained elusive. In this work, we studied the implication of polyamines during pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) in the model species Arabidopsis thaliana. Our data indicate that polyamines, particularly Put, accumulate in response to non-pathogenic Pseudomonas syringae pv. tomato DC3000 hrcC and in response to the purified PAMP flagellin22. Exogenously supplied Put to Arabidopsis seedlings induces defense responses compatible with PTI activation, such as callose deposition and transcriptional up-regulation of several PTI marker genes. Consistent with this, we show that Put primes for resistance against pathogenic bacteria. Through chemical and genetic approaches, we find that PTI-related transcriptional responses induced by Put are hydrogen peroxide and NADPH oxidase (RBOHD and RBOHF) dependent, thus suggesting that apoplastic ROS mediates Put signaling. Overall, our data indicate that Put amplifies PTI responses through ROS production, leading to enhanced disease resistance against bacterial pathogens

NLR Mutations Suppressing Immune Hybrid Incompatibility and Their Effects on Disease Resistance

Genetic divergence between populations can lead to reproductive isolation. Hybrid incompatibilities (HI) represent intermediate points along a continuum toward speciation. In plants, genetic variation in disease resistance (R) genes underlies several cases of HI. The progeny of a cross between Arabidopsis (Arabidopsis thaliana) accessions Landsberg erecta (Ler, Poland) and Kashmir2 (Kas2, central Asia) exhibits immune-related HI. This incompatibility is due to a genetic interaction between a cluster of eight TNL (TOLL/INTERLEUKIN1 RECEPTOR-NUCLEOTIDE BINDING-LEU RICH REPEAT) RPP1 (RECOGNITION OF PERONOSPORA PARASITICA1)-like genes (R1-R8) from Ler and central Asian alleles of a Strubbelig-family receptor-like kinase (SRF3) from Kas2. In characterizing mutants altered in Ler/Kas2 HI, we mapped multiple mutations to the RPP1-like Ler locus. Analysis of these suppressor of Ler/Kas2 incompatibility (sulki) mutants reveals complex, additive and epistatic interactions underlying RPP1-like Ler locus activity. The effects of these mutations were measured on basal defense, global gene expression, primary metabolism, and disease resistance to a local Hyaloperonospora arabidopsidis isolate (Hpa Gw) collected from Gorzów (Gw), where the Landsberg accession originated. Gene expression sectors and metabolic hallmarks identified for HI are both dependent and independent of RPP1-like Ler members. We establish that mutations suppressing immune-related Ler/Kas2 HI do not compromise resistance to Hpa Gw. QTL mapping analysis of Hpa Gw resistance point to RPP7 as the causal locus. This work provides insight into the complex genetic architecture of the RPP1-like Ler locus and immune-related HI in Arabidopsis and into the contributions of RPP1-like genes to HI and defense

Genome wide association mapping for the tolerance to the polyamine oxidase inhibitor guazatine in Arabidopsis thaliana

Guazatine is a potent inhibitor of polyamine oxidase (PAO) activity. In agriculture, guazatine is used as non-systemic contact fungicide efficient in the protection of cereals and citrus fruits against disease. The composition of guazatine is complex, mainly constituted by a mixture of synthetic guanidated polyamines (polyaminoguanidines). Here we have studied the effects from exposure to guazatine in the weed Arabidopsis thaliana. We report that micromolar concentrations of guazatine are sufficient to inhibit growth of Arabidopsis seedlings and induce chlorosis, whereas germination is barely affected. We observed the occurrence of quantitative variation in the response to guazatine between 107 randomly chosen Arabidopsis accessions. This enabled us to undertake genome-wide association (GWA) mapping that identified a locus on chromosome one associated with guazatine tolerance. CHLOROPHYLLASE 1 (CLH1) within this locus was studied as candidate gene, together with its paralog (CLH2). The analysis of independent clh1-2, clh1-3, clh2-3, clh2-2 and double clh1-2 clh2-3 mutant alleles indicated that CLH1 and/or CLH2 loss-of-function or expression down-regulation promote guazatine tolerance in Arabidopsis. We report a natural mechanism by which Arabidopsis populations can overcome toxicity by the fungicide guazatine

Putrescine elicits ROS-dependent activation of the salicylic acid pathway in Arabidopsis thaliana

Polyamines are small amines that accumulate during stress and contribute to disease resistance through as yet unknown signaling pathways. Using a comprehensive RNA-sequencing analysis, we show that early transcriptional responses triggered by each of the most abundant polyamines (putrescine, spermidine, spermine, thermospermine and cadaverine) exhibit specific quantitative differences, suggesting that polyamines (rather than downstream metabolites) elicit defense responses. Signaling by putrescine, which accumulates in response to bacteria that trigger effector triggered immunity (ETI) and systemic acquired resistance (SAR), is largely dependent on the accumulation of hydrogen peroxide, and is partly dependent on salicylic acid (SA), the expression of ENHANCED DISEASE SUSCEPTIBILITY (EDS1) and NONEXPRESSOR of PR GENES1 (NPR1). Putrescine elicits local SA accumulation as well as local and systemic transcriptional reprogramming that overlaps with SAR. Loss-of-function mutations in arginine decarboxylase 2 (ADC2), which is required for putrescine synthesis and copper amine oxidase (CuAO), which is involved in putrescine oxidation, compromise basal defenses, as well as putrescine and pathogen - triggered systemic resistance. These findings confirm that putrescine elicits ROS-dependent SA pathways in the activation of plant defenses

Pseudomonas germanica sp. nov., isolated from Iris germanica rhizomes

Through bacterial plant-endophyte extraction from rhizomes of Iris germanica plant, a Gram-stain-negative, aerobic, catalase- and oxidase-positive gammaproteobacterial strain, referred to as FIT28T, was isolated. FIT28T shows vigorous growth on nutrient rich media within the temperature range of 4-35 °C, with optimal growth at 28 °C, a wide pH tolerance from pH 5 to 11, and salt tolerance up to 6 % (w/v) NaCl. Colonies are white-yellow and quickly become mucoid. The results of analysis of the 16S rRNA gene sequence placed the strain within the genus Pseudomonas, and multilocus sequence analysis (MLSA) using 16S rRNA, rpoB, gyrB and rpoD concatenated sequences revealed that the closest relatives of FIT28T are Pseudomonas zeae OE48.2T, 'Pseudomonas crudilactis' UCMA 17988, Pseudomonas tensinigenes ZA5.3T, Pseudomonas helmanticensis OHA11T, Pseudomonas baetica a390T, Pseudomonas iridis P42T, Pseudomonas atagonensis PS14T and Pseudomonas koreensis Ps 9-14T, within the Pseudomonas koreensis subgroup of the Pseudomonas fluorescens lineage. The genome size of FIT28T is about 6.7 Mb with 59.09 mol% DNA G+C content. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) values calculated from the genomic sequences of FIT28T, and the closely related P. zeae OE48.2T are 95.23 and 63.4 %, respectively. Biochemical, metabolic and chemotaxonomic studies further support our proposal that Pseudomonas germanica sp. nov., should be considered a novel species of the genus Pseudomonas. Hence, the type strain FIT28T (=LMG 32353T=DSM 112698T) has been deposited in public cell-type culture centres

Tomato STEROL GLYCOSYLTRANSFERASE 1 silencing unveils a major role of steryl glycosides in plant and fruit development

Free and glycosylated sterols localize in the plant cell plasma membrane, where in combination with other lipids regulate its structure and function. The role of glycosylated sterols in regulating membrane-associated biological processes is more relevant in plants like tomato (Solanum lycopersicum), in which glycosylated sterols are the predominant sterols. A proper ratio of free sterols versus glycosylated sterols has proven to be essential for proper plant performance in several species, but almost nothing is known in tomato. To assess the role of glycosylated sterols in tomato plant and fruit development, we generated transgenic lines of tomato cultivar Micro-Tom expressing two different amiRNAs devised to silence STEROL GLYCOSYLTRANSFERASE 1, the most actively expressed of the four genes encoding sterol glycosyltransferases in this plant. STEROL GLYCOSYLTRANSFERASE 1 gene silencing caused moderate plant dwarfism and reduced fruit size. Analysis of the profile of glycosylated sterols throughout fruit development demonstrated that the maintenance of proper levels of these compounds during the early stages of fruit development is essential for normal fruit growth, since reduced levels of glycosylated sterols trigger a transcriptional downregulatory response that affects genes involved in processes that are critical for proper fruit development, such as seed filling, cell wall extension and auxin signaling

Pseudomonas fitomaticsae sp. nov., isolated at Marimurtra Botanical Garden in Blanes, Catalonia, Spain

In the framework of the research project called fitomatics, we have isolated and characterized a bacterial plant-endophyte from the rhizomes of Iris germanica, hereafter referred to as strain FIT81T. The bacterium is Gram negative, rod-shaped with lophotrichous flagella, and catalase- and oxidase-positive. The optimal growth temperature of strain FIT81T is 28 °C, although it can grow within a temperature range of 4-32 °C. The pH growth tolerance ranges between pH 5 and 10, and it tolerates 4% (w/v) NaCl. A 16S rRNA phylogenetic analysis positioned strain FIT81T within the genus Pseudomonas, and multilocus sequence analysis revealed that Pseudomonas gozinkensis IzPS32dT, Pseudomonas glycinae MS586T, Pseudomonas allokribbensis IzPS23T, 'Pseudomonas kribbensis' 46-2 and Pseudomonas koreensis PS9-14T are the top five most closely related species, which were selected for further genome-to-genome comparisons, as well as for physiological and chemotaxonomic characterization. The genome size of strain FIT81T is 6 492 796 base-pairs long, with 60.6 mol% of G+C content. Average nucleotide identity and digital DNA-DNA hybridization analyses yielded values of 93.6 and 56.1%, respectively, when the FIT81T genome was compared to that of the closest type strain P. gozinkensis IzPS32dT. Taken together, the obtained genomic, physiologic and chemotaxonomic data indicate that strain FIT81T is different from its closest relative species, which lead us to suggest that it is a novel species to be included in the list of type strains with the name Pseudomonas fitomaticsae sp. nov. (FIT81T=CECT 30374T=DSM 112699T)

Polyamine Oxidase 5 loss-of-function mutations in Arabidopsis thaliana trigger metabolic and transcriptional reprogramming and promote salt stress tolerance.

The family of polyamine oxidases (PAO) in Arabidopsis (AtPAO1-5) mediates polyamine (PA) back-conversion, which reverses the PA biosynthetic pathway from spermine, and its structural isomer thermospermine (tSpm), into spermidine and then putrescine. Here, we have studied the involvement of PA back-conversion in Arabidopsis salinity tolerance. AtPAO5 is the Arabidopsis PAO gene member most transcriptionally induced by salt stress. Two independent loss-of-function mutants (atpao5-2 and atpao5-3) were found to exhibit constitutively higher tSpm levels, with associated increased salt tolerance. Using global transcriptional and metabolomic analyses, the underlying mechanisms were studied. Stimulation of abscisic acid and jasmonates (JA) biosynthesis, and accumulation of important compatible solutes, such as sugars, polyols and proline, as well as TCA cycle intermediates were observed in atpao5 mutants under salt stress. Expression analyses indicate that tSpm modulates the transcript levels of several target genes, including many involved in the biosynthesis and signaling of JA, some of which are already known to promote salinity tolerance. Transcriptional modulation by tSpm is isomer-dependent, thus demonstrating the specificity of this response. Overall, we conclude that tSpm triggers metabolic and transcriptional reprogramming that promotes salt stress tolerance in Arabidopsis

Genetics of natural variation and environmental modulation of immune-related hybrid incompatibilities in Arabidopsis

[eng] Arabidopsis thaliana is a small ruderal weed with prominent north-hemisphere distribution. It has an autogamous style of reproduction, whereas about 3% of the natural population can exhibit outcrosses. Some of these outcrosses produce incompatible hybrids exhibiting temperature-dependent autoimmunity. The hybridization between accessions Ler (from North-Europe) and Kas-2 (from Central Asia) produces an incompatible hybrid. The genetics underlying the incompatibility is caused by the deleterious epistasic interactions between a unique Ler haplotype of RPP1-like genes and the SRF3 Kas-2 receptor-like kinase. The incompatibility exhibits recessive mendelian inheritance pattern and hybrids display strong dwarfism, spontaneous cell death, and sterility at 14 ◦C – 16 ◦C. By using EMS mutagenesis and CRISPR/Cas-9 gene editing on Ler/Kas-2 NIL plants, 15 RPP1-like intragenic loss-of-function mutations were identified. Furthermore, the impact of suppression of incompatibility was investigated in terms of disease resistance against hemibiotrophic or biotrophic pathogens. Moreover, changes on the global transcriptome and metabolite profiling were studied. The contribution of individual RPP1-like genes for the recognition of a local Hpa isolate (Gw) was carried out by cloning ATR1 effector and performing transient expression assays in tobacco plants. The environmental modulation of autoimmunity was studied with a focus on the nitrogen-based mineral nutrition. We found that increasing ammonium was sufficient to suppress the incompatibility and the mechanism behind implies signaling through nitric oxide and the contribution of the systemic acquired response master gene, NPR1. Furthermore, we suggest that ammonium enriched media has an impact on resistance to Pst turning the enhanced resistance on incompatible hybrids down to the parental lines. Finally, we investigated the natural variation to the plant herbicide, guazatine, on a set of 107 accessions. We found an association for the chlorophyllase 1 gene which was selected for functional validation together with the paralog chlorophyllase 2 gene. Chlorophyllase individual mutants and double mutants exhibit increased tolerance to guazatine. No geographical pattern between the tolerant genotype and gene phylogeny was found, whereas high proportion of tolerant accessions was detected in populations for Germany.[cat] Arabidopsis thaliana és una planta ruderal petita amb una distribució prominent a l'hemisferi nord. Té una reproducció del tipus autogàmic. No obstant, en la natura, es poden donar creuaments amb una freqüència pel voltant del 3%. Alguns d’aquests creuaments, poden originar híbrids incompatibles amb resposta autoimmunitària que és dependent de la temperatura ambiental. La hibridació entre els accessions Ler (Europa del Nord) i Kas-2 (Àsia Central) produeix un híbrid incompatible. La genètica subjacent a la incompatibilitat és causada per les interaccions epistàsiques deletèries donades per la presència de l’haplotip Ler RPP1-like i el receptor quinasa de Kas-2, SRF3. Aquesta incompatibilitat té un patró d'herència recessiva i els híbrids són nans, mostren mort cel·lular espontània i esterilitat a 14ºC – 16ºC. Mitjançant la mutagènesi per EMS i edició gènica per CRISPR/Cas-9 en Ler / Kas-2 NIL, s’han identificat 15 mutacions intragèniques en l’haplotip RPP1-like. Es va investigar l'impacte de la supressió de la incompatibilitat en termes de resistència a patògens hemibiotròfics o biotròfics. Tanmateix, es va fer un estudi del metaboloma i transcriptoma global per tal de veure l’impacte de la autoimmunitat en el metabolisme. La contribució de gens RPP1-like pel reconeixement del patogen local Hpa (Gw) s’ha investigat mitjançant la clonació d’efector ATR1 i assajos de coinfiltració en sistema heteròleg. També es va estudiar la modulació ambiental sobre l’autoimmunitat en aspectes nutricionals. S’ha trobat que un augment d’amoni era suficient per suprimir la incompatibilitat híbrida i hem suggerit la contribució moduladora per NO i el gen NPR1. Finalment, hem investigat la variació natural per fungicida i herbicida, guazatina, en 107 accessions. Hem trobat una associació pel gen de la clorofilasa 1 que va ser seleccionada per a la seva validació funcional, juntament amb el gen paràleg de la clorofilasa 2. Els mutants individuals de clorofilasa i els dobles mutants, van mostra una major tolerància a la guazatina tan a nivell de contigut de clorofil·les com creixement de l’arrel. No s’ha trobat cap patró geogràfic entre els genotips tolerants i la filogènia dels gens. No obstant, una gran proporció d’accessions tolerants es va detectar en les poblacions d'Alemanya