Factores de Virulencia conservados en bacterias fitopatógenas alteran el metabolismo del azufre en plantas durante la interacción planta-patógeno

En los últimos años se ha progresado ampliamente en el conocimiento de la percepción de patógenos por parte de las plantas y su consecuente activación de señalización inmune. En este sentido, la utilización de efectores -proteínas de patógenos que actúan como factores de virulencia-, como sondas ha sido fundamental para la identificación de los componentes moleculares que inician la respuesta inmune. Las bacterias fitopatógenas Ralstonia solanacearum y Pseudomonas syringae son dos de las bacterias más relevantes a nivel agronómico y académico debido a sus amplios rangos de hospedador y a las grandes pérdidas que causan en diferentes cultivos. Los ciclos de vida y nichos de infección de R. solanacearum y P. syringae presentan importantes diferencias. Sin embargo, entre el amplio arsenal de efectores que codifican, hay un pequeño número de efectores de R. solanacearum que presentan ortólogos en P. syringae. El objetivo de este trabajo es el estudio de estos efectores conservados, que pueden tener un potencial impacto durante la interacción planta-patógeno. Uno de estos efectores, Rip15, es esencial para la virulencia de R. solanacearum en tomate. Un análisis transcriptómico de plantas transgénicas de Arabidopsis thaliana expresando constitutivamente Rip15 mostró que Rip15 induce una respuesta relacionada con la deficiencia de azufre en planta. P. syringae pv. phaseolicola, el agente causal de la grasa de la judía, presenta un efector ortólogo a Rip15, llamado HopAV. En este trabajo presentamos las posibles dianas moleculares de Rip15/HopAV así como la relevancia de la alteración del metabolismo del azufre en la interacción planta-patógeno.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The bacterial effector HopZ1a acetylates MKK7 to suppress plant immunity

The Pseudomonas syringae type III secretion system translocates effector proteins into the host cell cytosol to suppress plant basal immunity. Effector HopZ1a suppresses local and sys- temic immunity triggered by pathogen-associated molecular patterns (PAMPs) and effectors, through target acetylation. HopZ1a has been shown to target several plant proteins, but none fully substantiates HopZ1a-associated immune suppression. Here, we investigate Arabidopsis thaliana mitogen-activated protein kinase kinases (MKKs) as potential targets, focusing on AtMKK7, a positive regulator of local and systemic immunity. We analyse HopZ1a interference with AtMKK7 by translocation of HopZ1a from bacteria inoculated into Arabidopsis expressing MKK7 from an inducible promoter. Reciprocal pheno- types are analysed on plants expressing a construct quenching MKK7 native expression. We analyse HopZ1a–MKK7 interaction by three independent methods, and the relevance of acetylation by in vitro kinase and in planta functional assays. We demonstrate the AtMKK7 contribution to immune signalling showing MKK7- dependent flg22-induced reactive oxygen species (ROS) burst, MAP kinas (MAPK) activation and callose deposition, plus AvrRpt2-triggered MKK7-dependent signalling. Furthermore, we demonstrate HopZ1a suppression of all MKK7-dependent responses, HopZ1a–MKK7 interac- tion in planta and HopZ1a acetylation of MKK7 with a lysine required for full kinase activity. We demonstrate that HopZ1a targets AtMKK7 to suppress local and systemic plant immunity.RTI2018- 095069-B-I00, BIO2015-64391-R, UMA18-FEDERJA-070, FPU14/04233, XDB27040204, Universidad de Málaga-CBU

Dynamic expression of Ralstonia solanacearum virulence factors and metabolism-controlling genes during plant infection

Altres ajuts: CERCA Programme/Generalitat de Catalunya. P. Sebastià received the support of a fellowship (code is LCF/BQ/IN17/11620004) from la Caixa Foundation (identifier [ID] 100010434)Background: Ralstonia solanacearum is the causal agent of bacterial wilt, a devastating plant disease responsible for serious economic losses especially on potato, tomato, and other solanaceous plant species in temperate countries. In R. solanacearum, gene expression analysis has been key to unravel many virulence determinants as well as their regulatory networks. However, most of these assays have been performed using either bacteria grown in minimal medium or in planta, after symptom onset, which occurs at late stages of colonization. Thus, little is known about the genetic program that coordinates virulence gene expression and metabolic adaptation along the different stages of plant infection by R. solanacearum. Results: We performed an RNA-sequencing analysis of the transcriptome of bacteria recovered from potato apoplast and from the xylem of asymptomatic or wilted potato plants, which correspond to three different conditions (Apoplast, Early and Late xylem). Our results show dynamic expression of metabolism-controlling genes and virulence factors during parasitic growth inside the plant. Flagellar motility genes were especially up-regulated in the apoplast and twitching motility genes showed a more sustained expression in planta regardless of the condition. Xylem-induced genes included virulence genes, such as the type III secretion system (T3SS) and most of its related effectors and nitrogen utilisation genes. The upstream regulators of the T3SS were exclusively up-regulated in the apoplast, preceding the induction of their downstream targets. Finally, a large subset of genes involved in central metabolism was exclusively down-regulated in the xylem at late infection stages. Conclusions: This is the first report describing R. solanacearum dynamic transcriptional changes within the plant during infection. Our data define four main genetic programmes that define gene pathogen physiology during plant colonisation. The described expression of virulence genes, which might reflect bacterial states in different infection stages, provides key information on the R. solanacearum potato infection process

Pseudomonas syringae Differentiates into Phenotypically Distinct Subpopulations During Colonization of a Plant 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.Ministerio de Ciencia e Innovación BIO2009-11516, AGL2010-22287-C02-2Ministerio de Economía y Competitividad BIO2012-35641, BIO2015-64391-

Suppression of HopZ Effector-Triggered Plant Immunity in a Natural Pathosystem

Many type III-secreted effectors suppress plant defenses, but can also activate effector-triggered immunity (ETI) in resistant backgrounds. ETI suppression has been shown for a number of type III effectors (T3Es) and ETI-suppressing effectors are considered part of the arms race model for the co-evolution of bacterial virulence and plant defense. However, ETI suppression activities have been shown mostly between effectors not being naturally expressed within the same strain. Furthermore, evolution of effector families is rarely explained taking into account that selective pressure against ETI-triggering effectors may be compensated by ETI-suppressing effector(s) translocated by the same strain. The HopZ effector family is one of the most diverse, displaying a high rate of loss and gain of alleles, which reflects opposing selective pressures. HopZ effectors trigger defense responses in a variety of crops and some have been shown to suppress different plant defenses. Mutational changes in the sequence of ETI-triggering effectors have been proposed to result in the avoidance of detection by their respective hosts, in a process called pathoadaptation. We analyze how deleting or overexpressing HopZ1a and HopZ3 affects virulence of HopZ-encoding and non-encoding strains. We find that both effectors trigger immunity in their plant hosts only when delivered from heterologous strains, while immunity is suppressed when delivered from their native strains. We carried out screens aimed at identifying the determinant(s) suppressing HopZ1a-triggered and HopZ3-triggered immunity within their native strains, and identified several effectors displaying suppression of HopZ3-triggered immunity. We propose effector-mediated cross-suppression of ETI as an additional force driving evolution of the HopZ family

The transcriptional regulator JAZ8 interacts with the C2 protein from geminiviruses and limits the geminiviral infection in Arabidopsis

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signalling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favours geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants do not display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

The transcriptional regulator JAZ8 interactswith the C2 protein from geminivirusesand limits the geminiviral infection in Arabidopsis

Jasmonates (JAs) are phytohormones that finely regulate critical biological processes, including plant development and defense. JASMONATE ZIM-DOMAIN (JAZ) proteins are crucial transcriptional regulators that keep JA-responsive genes in a repressed state. In the presence of JA-Ile, JAZ repressors are ubiquitinated and targeted for degradation by the ubiquitin/proteasome system, allowing the activation of downstream transcription factors and, consequently, the induction of JA-responsive genes. A growing body of evidence has shown that JA signaling is crucial in defending against plant viruses and their insect vectors. Here, we describe the interaction of C2 proteins from two tomato-infecting geminiviruses from the genus Begomovirus, tomato yellow leaf curl virus (TYLCV) and tomato yellow curl Sardinia virus (TYLCSaV), with the transcriptional repressor JAZ8 from Arabidopsis thaliana and its closest orthologue in tomato, SlJAZ9. Both JAZ and C2 proteins colocalize in the nucleus, forming discrete nuclear speckles. Overexpression of JAZ8 did not lead to altered responses to TYLCV infection in Arabidopsis; however, knock-down of JAZ8 favors geminiviral infection. Low levels of JAZ8 likely affect the viral infection specifically, since JAZ8-silenced plants neither display obvious developmental phenotypes nor present differences in their interaction with the viral insect vector. In summary, our results show that the geminivirus-encoded C2 interacts with JAZ8 in the nucleus, and suggest that this plant protein exerts an anti-geminiviral effect.This work was supported by the Spanish Ministerio de Ciencia y Tecnología (PID2019-107657RB-C22) (ER-B), FEDER program (UMA20-FEDERJA-021) (AG-C), the Shanghai Center for Plant Stress Biology, the Chinese Academy of Sciences, and the Federal Ministry of Education and Research (BMBF) and the Baden-Württemberg Ministry of Science as part of the Excellence Strategy of the German Federal and State Governments (RL-D). TR-D was supported by a President's International Fellowship Initiative (PIFI) postdoctoral fellowship (No. 2016PB042) from the Chinese Academy of Sciences, the “Programa Juan de la Cierva” (IJCI-2017-33367) from the MCIN and FEDER program UMA20-FEDERJA-132 by AEI and by “ERDF A way of making Europe,” by the “European Union.” Funding for Open Access charge: Universidad de Málaga / CBUA

Solution of the Navier–Stokes Equations for the Processes of Inertial Gas Dynamic Separation in the Curvilinear Channels

Аналіз результатів показав, що вісесимметричний газовий потік рідини уздовж вигнутого каналу утворює періодичні вихрі в порожнинах по зовнішньому радіусу, а також пульсації динамічного тиску. Подальші дослідження будуть спрямовані на чисельне моделювання газодинамічної сепарації в криволінійних каналах з гнучкими стінками.У результаті аналітичного розв'язання рівнянь Нав'є-Стокса для потоку газу в плоскому напівкруглому кільцевому каналі визначені радіальна і окружна компоненти швидкості з урахуванням граничних умов, обмежень і гіпотез. Отримані рівняння для визначення витрат газу і вираз для розподілу тиску.В результате аналитического решения уравнений Навье-Стокса для потока газа в плоском полукруглом кольцевом канале определены радиальная и окружная компоненты скорости с учетом граничных условий, ограничений и гипотез. Получены уравнения для определения расхода газа и выражение для распределения давления.In this paper as a result of the analytical solution of the Navier-Stokes equations for gas flow in the plane semicircular annular channel the radial and circumference velocity components were determined taking into account boundary conditions, limitations and hypotheses. Equation for gas leakages determination and expression for pressure distribution were received

Genetic Analysis of the Individual Contribution to Virulence of the Type III Effector Inventory of Pseudomonas syringae pv. phaseolicola

Several reports have recently contributed to determine the effector inventory of the sequenced strain Pseudomonas syringae pv. phaseolicola (Pph) 1448a. However, the contribution to virulence of most of these effectors remains to be established. Genetic analysis of the contribution to virulence of individual P. syringae effectors has been traditionally hindered by the lack of phenotypes of the corresponding knockout mutants, largely attributed to a high degree of functional redundancy within their effector inventories. In support of this notion, effectors from Pseudomonas syringae pv. tomato (Pto) DC3000 have been classified into redundant effector groups (REGs), analysing virulence of polymutants in the model plant Nicotiana benthamiana. However, using competitive index (CI) as a virulence assay, we were able to establish the individual contribution of AvrPto1PtoDC3000 to Pto DC3000 virulence in tomato, its natural host, even though typically, contribution to virulence of AvrPto1 is only shown in strains also lacking AvrPtoB (also called HopAB2), a member of its REG. This report raised the possibility that even effectors targeting the same defence signalling pathway may have an individual contribution to virulence, and pointed out to CI assays as the means to establish such a contribution for individual effectors. In this work, we have analysed the individual contribution to virulence of the majority of previously uncharacterised Pph 1448a effectors, by monitoring the development of disease symptoms and determining the CI of single knockout mutants at different stages of growth within bean, its natural host. Despite their potential functional redundancy, we have found individual contributions to virulence for six out of the fifteen effectors analysed. In addition, we have analysed the functional relationships between effectors displaying individual contribution to virulence, highlighting the diversity that these relationships may present, and the interest of analysing their functions within the context of the infection

Synaptotagmins at the endoplasmic reticulum-plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

Endoplasmic Reticulum-Plasma Membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to WT while the levels of most glycerolipid species remain unchanged. Additionally, SYT1-GFP preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress