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Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses., Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses., Plants use different receptors to detect potential pathogens: membrane-anchored pattern recognition receptors (PRRs) activated upon perception of pathogen-associated molecular patterns (PAMPs) that elicit pattern-triggered immunity (PTI); and intracellular nucleotide-binding leucine-rich repeat proteins (NLRs) activated by detection of pathogen-derived effectors, activating effector-triggered immunity (ETI). The interconnections between PTI and ETI responses have been increasingly reported. Elevated NLR levels may cause autoimmunity, with symptoms ranging from fitness cost to developmental arrest, sometimes combined with run-away cell death, making accurate control of NLR dosage key for plant survival. Small RNA-mediated gene regulation has emerged as a major mechanism of control of NLR dosage. Twenty-two nucleotide miRNAs with the unique ability to trigger secondary siRNA production from target transcripts are particularly prevalent in NLR regulation. They enhance repression of the primary NLR target, but also bring about repression of NLRs only complementary to secondary siRNAs. We summarize current knowledge on miRNAs and siRNAs in the regulation of NLR expression with an emphasis on 22 nt miRNAs and propose that miRNA and siRNA regulation of NLR levels provides additional links between PTI and NLR defense pathways to increase plant responsiveness against a broad spectrum of pathogens and control an efficient deployment of defenses.

Plants possess a battery of dedicated intracellular immune receptors known as nucleotide-binding leucine-rich repeat proteins (NLRs) involved in recognition of pathogen derived efectors and activation of efector-triggered immunity (ETI). In a recent work, we report a two-tiered regulatory network in Arabidopsis mediated by a microRNA (miR825-5p) and a wave of secondary NLRderived phased small RNAs (phasi-NLRs) involved in silencing dozens of NLR genes encoding Toll/interleukin-1 NLRs (TNLs). In our model, targeting of MIST1 (microRNA-silenced TNL1) transcripts by miR825-5p, triggers the generation of phasi-NLRs that subsequently silence a wide network of TNL encoding genes and reinforce the silencing of MIST1 (López-Márquez et al., 2021). Although miRNA and phasi-NLR mediated regulation of NLRs has been characterized to some extent, the current knowledge regarding how these miRNA-target-phasiRNA networks are modulated and their contribution to the immune response during diferent developmental stages is still scarce (Deng et al., 2018; Cui et al., 2019). Thus, through generation of GUS-based A. thaliana reporter lines we have characterized the expression pattern of both genes (MIR825 and MIST1) as well as the domain activity of miR825-5p. Moreover, a comprehensive analysis of RNA and small RNA-seq datasets, as well as the use of diferent orthogonal approaches, unravelled a hitherto unknow connection between miR825-5p/phasi-NLR mediated regulation of NLRs and plant development. Further analysis also supports the notion that the action of this regulatory mechanism is restricted to control NLR expression in an organ specifc way, mainly operating in leaves of the model plant. At present, we are working to understand how the changes on miRNA/phasi-NLR levels during developmental stages modulate the strength of the immune response and the biological impact of this developmental-dependent regulation on plant health. In summary, our results point to a developmentally regulat

Minimally processed or fresh fruits and vegetables are unfortunately linked to an increasing number of food-borne diseases, such as salmonellosis. One of the relevant virulence factors during the initial phases of the infection process is the bacterial flagellum. Although its function is well studied in animal systems, contradictory results have been published regarding its role during plant colonization. In this study, we tested the hypothesis that Salmonella’s flagellin plays a versatile function during the colonization of tomato plants. We have assessed the persistence in plant tissues of a Salmonella enterica wild type strain, and of a strain lacking the two flagellins, FljB and FliC.

Olive trees are known for their capacity to adapt to drought through several phenotypic and molecular variations, although this can vary according to the different provenances of the same olive cultivar. We confronted the same olive cultivar from two different location in Spain: Freila, in the Granada province, with low annual precipitation, and Grazalema, in the Cadiz province, with high annual precipitation, and subjected them to five weeks of severe drought stress. We found distinctive physiological and developmental adaptations among the two provenances. Thus, trees from Freila subjected to drought stress exhibited increasing root dry weights and decreasing leaf numbers and relative stem heights. On the other hand, the treatment with drought in Grazalema trees reduced their leaf chlorophyll contents, but increased their relative stem diameter and their root hydraulic conductivity. The physiological responses of Freila tree roots to drought were linked to different molecular adaptations that involved the regulation of genes related to transcription factors induced by ABA, auxin and ethylene signaling, as well as, the action of a predicted membrane intrinsic protein (MIP). On the other hand, the responses of Grazalema trees were related with different root genes related to oxidation-reduction, ATP synthesis, transduction and posttranslational regulation, with a special mention to the cytokinins signaling through the transcript predicted as a histidine-containing phosphotransfer protein. Our results show that olive trees adapted to dry environments will adjust their growth and water uptake capacity through transcription factors regulation, and this will influence the different physiological responses to drought stress.

Plants encode numerous intracellular receptors known as nucleotide-binding leucine-rich repeat receptors (NLRs) that recognize pathogen-derived effectors or their activity to activate defenses. miRNAs regulate NLR genes in many species, often triggering the production of phased siRNAs (phasiRNAs). Most such examples involve genes encoding NLRs carrying coiled-coil domains, although a few include genes encoding NLRs carrying a Toll/interleukin-1 domain (TNL). Here, we characterize the role of miR825-5p in Arabidopsis, using a combination of bioinformatics, transgenic plants with altered miRNA levels and/or reporters, small RNAs, and virulence assays. We demonstrate that miR825-5p down-regulates the TNL MIST1 by targeting for endonucleolytic cleavage the sequence coding for TIR2, a highly conserved amino acid motif, linked to a catalytic residue essential for immune function. miR825-5p acts as a negative regulator of basal resistance against Pseudomonas syringae. miR825-5p triggers the production from MIST1 of a large number of phasiRNAs that can mediate cleavage of both MIST1 and additional TNL gene transcripts, potentially acting as a regulatory hub. miR825-5p is expressed in unchallenged leaves and transcriptionally down-regulated in response to pathogen-associated molecular patterns (PAMPs). Our results show that miR825-5p, which is required for full expression of PAMP-triggered immunity, establishes a link between PAMP perception and expression of uncharacterized TNL genes.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.

Bacterial microcolonies with heterogeneous sizes are formed during colonization of Phaseolus vulgaris by Pseudomonas syringae. Heterogeneous expression of structural and regulatory components of the P. syringae type III secretion system (T3SS), essential for colonization of the host apoplast and disease development, is likewise detected within the plant apoplast. T3SS expression is bistable in the homogeneous environment of nutrient-limited T3SS-inducing medium, suggesting that subpopulation formation is not a response to different environmental cues. T3SS bistability is reversible, indicating a non-genetic origin, and the T3SSHIGH and T3SSLOW subpopulations show differences in virulence. T3SS bistability requires the transcriptional activator HrpL, the double negative regulatory loop established by HrpV and HrpG, and may be enhanced through a positive feedback loop involving HrpA, the main component of the T3SS pilus. To our knowledge, this is the first example of phenotypic heterogeneity in the expression of virulence determinants during colonization of a non-mammalian host.

Plant architecture is a critical trait in fruit crops that can significantly influence yield, pruning, planting density and harvesting. Little is known about how plant architecture is genetically determined in olive, were most of the existing varieties are traditional with an architecture poorly suited for modern growing and harvesting systems. In the present study, we have carried out microarray analysis of meristematic tissue to compare expression profiles of olive varieties displaying differences in architecture, as well as seedlings from their cross pooled on the basis of their sharing architecture-related phenotypes. The microarray used, previously developed by our group has already been applied to identify candidates genes involved in regulating juvenile to adult transition in the shoot apex of seedlings. Varieties with distinct architecture phenotypes and individuals from segregating progenies displaying opposite architecture features were used to link phenotype to expression. Here, we identify 2252 differentially expressed genes (DEGs) associated to differences in plant architecture. Microarray results were validated by quantitative RT-PCR carried out on genes with functional annotation likely related to plant architecture. Twelve of these genes were further analyzed in individual seedlings of the corresponding pool. We also examined Arabidopsis mutants in putative orthologs of these targeted candidate genes, finding altered architecture for most of them. This supports a functional conservation between species and potential biological relevance of the candidate genes identified. This study is the first to identify genes associated to plant architecture in olive, and the results obtained could be of great help in future programs aimed at selecting phenotypes adapted to modern cultivation practices in this species.

The adaptation capacity of olive trees to different environments is well recognized. However, the presence of microorganisms in the soil is also a key factor in the response of these trees to drought. The objective of the present study was to elucidate the effects of different arbuscular mycorrhizal (AM) fungi coming from diverse soils on olive plant growth and water relations. Olive plants were inoculated with native AM fungal populations from two contrasting environments, that is, semi-arid – Freila (FL) and humid – Grazalema (GZ) regions, and subjected to drought stress. Results showed that plants grew better on GZ soil inoculated with GZ fungi, indicating a preference of AM fungi for their corresponding soil. Furthermore, under these conditions, the highest AM fungal diversity was found. However, the highest root hydraulic conductivity (Lp) value was achieved by plants inoculated with GZ fungi and growing in FL soil under drought conditions. So, this AM inoculum also functioned in soils from different origins. Nine novel aquaporin genes were also cloned from olive roots. Diverse correlation and association values were found among different aquaporin expressions and abundances and Lp, indicating how the interaction of different aquaporins may render diverse Lp values.

The Pseudomonas syringae type III-secreted effector HopZ1a is a member of the HopZ/YopJ superfamily of effectors that triggers immunity in Arabidopsis. We have previously shown that HopZ1a suppresses both local [effector-triggered immunity (ETI)] and systemic immunity [systemic acquired resistance (SAR)] triggered by the heterologous effector AvrRpt2. HopZ1a has been shown to possess acetyltransferase activity, and this activity is essential to trigger immunity in Arabidopsis. HopZ1a acetyltransferase activity has been reported to require the auto-acetylation of the effector on a specific lysine (K289) residue. In this paper we analyze the relevance of autoacetylation of lysine residue 289 in HopZ1a ability to suppress plant defenses, and on the light of the results obtained, we also revise its relevance for HopZ1a avirulence activity. Our results indicate that, while the HopZ1aK289R mutant is impaired to some degree in its virulence and avirulence activities, is by no means phenotypically equivalent to the catalytically inactive HopZ1aC216A, since it is still able to trigger a defense response that induces detectable macroscopic HR and effectively protects Arabidopsis from infection, reducing growth of P. syringae within the plant. We also present evidence that the HopZ1aK289R mutant still displays virulence activities, partially suppressing both ETI and SAR.

Plant disease epidemics result from interactions of a susceptible host, a virulent pathogen and conducive environment. Shifts in any component can change disease expression to still unknown directions. Verticillium wilt (VW) of olive caused by the soilborne fungus Verticillium dahliae (Vd) is of major concern for olive industry in the Mediterranean basin. We carried out experiments using olive cvs. Picual and Arbequina, which grew in soil infested by the defoliating (D) or non-defoliating (ND) Vd pathotypes at 20, 24 and 28ºC and CO2 levels of 386, 550 and 750 ppm, representative of current and future SRES-IPCC A2 and B2 scenarios for southern Spain. Surface response models quantified the combined effects of temperature and CO2 on VW intensity and plant growth, demonstrating a differential effect of biotic and abiotic factors. Optimum VW development occurred at 20-24°C and current CO2, being faster and severe in 'Picual'/D. Raising CO2 delayed VW and Vd infection. The transcriptomic profile of olive cultivars in response to Vd infection and the three climate SRES scenarios was assessed using a 12-plex microarray of 37,449 olive unigenes (OLEAGEN project). Main differences in the expression profiles were due to olive genotype, followed by the climatic conditions at the three SRES scenarios and in a lesser extent by Vd infection. Thus, plants growing at current climate showed higher number of differentially expressed genes compared to plants growing at SRES-IPCC A2 and B2 scenarios with a general trend to decrease the number of significantly transcribed genes as temperature and CO2 increased. Furthermore, a higher number of genes (up to 10x) were expressed differentially in 'Arbequina' than in 'Picual' in response to environmental changes as well as to infection by the D pathotype with a low number of genes being common for both cultivars.

La temperatura y la concentración de dióxido de carbono (CO2) atmosférico son las variables ambientales asociadas al Cambio Climático que mayor impacto pueden ejercer sobre los patosistemas agrícolas, modificando hacia direcciones aún desconocidas la virulencia de los patógenos, así como el nivel de resistencia de las plantas a éstos. En este trabajo se ha realizado un estudio transcriptómico utilizando un microarray 12-plex 135K de 37.449 unigenes de olivo (Proyecto OLEAGEN) para determinar el efecto de la temperatura y CO2 ambiental sobre el desarrollo de la Verticilosis, y su interacción con el nivel de virulencia de patotipos de Verticllium dahliae (VD) y la susceptibilidad del genotipo de olivo en momentos previos a la aparición de síntomas. Para ello plantones de olivo de los cvs. Picual y Arbequina, crecieron en suelo infestado por cada uno de los patotipos defoliante (D) y no defoliante (ND) de VD, con condiciones de temperatura y CO2 representativas de los niveles actuales ambientales de ambos factores (20ºC, 386 ppm) así como sus estimaciones por modelos globales de simulación de clima para los escenarios AR4 del ICPP para el sur de España B2 (24ºC, 550 ppm) y A2 (28ºC, 750 ppm). Los resultados mostraron que, en las condiciones evaluadas, las principales diferencias en los perfiles de expresión son debidas al genotipo de olivo, seguidas de las condiciones ambientales y la infección por VD. Así, en plantas control (no inoculadas) se observaron grandes diferencias (P<0.001; 8-fold) en los perfiles de expresión de ‘Arbequina’ frente ‘Picual’ que se acentuaron en los escenarios B2 a A2, en ese orden, con un 20% de genes con expresión diferencial entre ambos cultivares expresándose en los tres escenarios climáticos, mientras que estas diferencias se atenuaron en plantas infectadas por el patotipo D. Por otro lado, el número de genes que se expresaron diferencialmente (P<0.001; 2-fold) en ‘Arbequina’ fue muy superior al observado en ‘Picual’ tanto en respuesta a

La temperatura y la concentración de dióxido de carbono (CO2) atmosférico son las variables ambientales asociadas al Cambio Climático que mayor impacto pueden ejercer sobre los patosistemas agrícolas, modificando hacia direcciones aún desconocidas la virulencia de los patógenos, así como el nivel de resistencia de las plantas a éstos . En este trabajo se ha realizado un estudio transcriptómico utilizando un microarray 12-plex 135K de 37 .449 unigenes de olivo (Proyecto OLEAGEN) para determinar el efecto de la temperatura y CO2 ambiental sobre el desarrollo de la Verticilosis, y su interacción con el nivel de virulencia de patotipos de Verticllium dahliae (VD) y la susceptibilidad del genotipo de olivo en momentos previos a la aparición de síntomas . Para ello plantones de olivo de los cvs . Picual y Arbequina, crecieron en suelo infestado por cada uno de los patotipos defoliante (D) y no defoliante (ND) de VD, con condiciones de temperatura y CO2 representativas de los niveles actuales ambientales de ambos factores (20ºC, 386 ppm) así como sus estimaciones por modelos globales de simulación de clima para los escenarios AR4 del ICPP para el sur de España B2 (24ºC, 550 ppm) y A2 (28ºC, 750 ppm) . Los resultados mostraron que, en las condiciones evaluadas, las principales diferencias en los perfiles de expresión son debidas al genotipo de olivo, seguidas de las condiciones ambientales y la infección por VD . Así, en plantas control (no inoculadas) se observaron grandes diferencias (P<0 .001; 8-fold) en los perfiles de expresión de ‘Arbequina’ frente ‘Picual’ que se acentuaron en los escenarios B2 a A2, en ese orden, con un 20% de genes con expresión diferencial entre ambos cultivares expresándose en los tres escenarios climáticos, mientras que estas diferencias se atenuaron en plantas infectadas por el patotipo D . Por otro lado, el número de genes que se expresaron diferencialmente (P<0 .001; 2-fold) en ‘Arbequina’ fue muy superior al observado en ‘Picual’ tanto en re

IS 200 is a mobile element found in a variety of eubacterial genera, such as Salmonella, Escherichia, Shigella, Vibrio, Enterococcus, Clostridium, Helicobacter, and Actinobacillus. In addition, IS200-like elements are found in archaea. IS 200 elements are very small (707–711 bp) and contain a single gene. Cladograms constructed with IS200 DNA sequences suggest that IS 200 has not spread among eubacteria by horizontal transfer; thus it may be an ancestral component of the bacterial genome. Self-restraint may have favored this evolutionary endurance; in fact, unlike typical mobile elements, IS 200 transposes rarely. Tight repression of transposase synthesis is achieved by a combination of mechanisms: inefficient transcription, protection from impinging transcription by a transcriptional terminator, and repression of translation by a stem-loop mRNA structure. A consequence of IS 200 self-restraint is that the number and distribution of IS 200