hrpZ_(Psg12) Gene of Pseudomonas syringae pv.glycinea can Enhance Pathogenicity of the Pathogen on Soybean and Cause the Hypersensitive Response of Tobacco

[ Objective ] The paper was to confirrm the effect of hrpZpsg12 gene on the pathogenicity of Pseudomonas syringae pv. glycinea. [ Method ] hrpZpsg12 gene was cloned from P. syringae using PCR method. The knockout plasmid pKNOCK-Cm with suicide characteristics and cosmid pUFR034 with complementation func- tion were used to construct the mutation vector pKNOCK477-7 and complementary vector pUFR1026-68 of hrpZpsg12 gene, the mutant 477-1 and the functional com- plementation unit 1026-5 of the gene was also screened out. Three strains including wild-type Psg12, mutant 477-1 and complementary unit 1026-5 were simultane- ously inoculated into soybean leaves and tobacco leaves, then pathogenicity determination and hypersensitive reaction analysis were carried out. [ Result] All the inoculated leaves of soybean and tobacco produced reaction lesion. However, the sizes of reaction lesion were different. The lesion in the leaves inoculated with Psgl2 was relatively large, while the lesion in the leaves inoculated with 477-1 was relatively small; the lesion of complementary unit 1026-5 was similar to wild- type Psgl2. Analysis of reproduction quantity of bacteria in lesions showed that the reproduction quantity of wild-type Psg12 was the highest, while that of mutant 477-1 was the lowest. The reproduction quantity of complementary unit 1026-5 was similar to that of wild-type Psg12. [ Conclusion] hrpZpsg12 gene could enhance the pathogenicity of P. syrimgae on Soybean and produce hypersensitive response in tobacco.

Functional identification of phenazine biosynthesis genes in plant pathogenic bacteria Pseudomonas syringae pv. tomato and Xanthomonas oryzae pv. oryzae

Phenazines are secondary metabolites with broad spectrum antibiotic activity and thus show high potential in biological control of pathogens. In this study, we identified phenazine biosynthesis （phz） genes in two genome-completed plant pathogenic bacteria Pseudomonas syringae pv. tomato （Pst） DC3000 and Xanthomonas oryzae pv. oryzae （Xoo） PXO99A. Unlike the phz genes in typical phenazine-producing pseudomonads, phz homologs in Pst DC3000 and Xoo PXO99A consisted of phzC/D/E/F/G and phzC/E1/E2/F/G, respectively, and the both were not organized into an operon. Detection experiments demonstrated that phenazine-l-carboxylic acid （PCA） of Pst DC3000 accumulated to 13.4 IJg L-1, while that of Xoo PXO99A was almost undetectable. Moreover, Pst DC3000 was resistant to 1 mg mL-1 PCA, while Xoo PXO99A was sensitive to 50 IJg mL ~ PCA. Furthermore, mutation of phzF blocked the PCA production and significantly reduced the pathogenicity of Pst DC3000 in tomato, while the complementary strains restored these phenotypes. These results revealed that Pst DC3000 produces low level of and is resistant to phenazines and thus is unable to be biologically controlled by phenazines. Additionally, phz-mediated PCA production is required for full pathogenicity of Pst DC3000. To our knowledge, this is the first report of PCA production and its function in pathogenicity of a plant pathogenic P. syringae strain.

Pear Blossom Blast Caused by Pseudomonas syringae pv. syringae in China

This study was done to determine the causal organism of the pear blossom and bud blast in China. It was identified by a bacteriological test, electro-microscopic observation, Koch＇s postulate test, Biolog, fatty acid methyl esters （FAMEs）, and a polymerase chain reaction （PCR） test, and compared with the standard reference strains. Six representative strains out of 20 pathogenic bacterial isolates from 16 diseased samples showed characteristics similar to three standard strains of Pseudomonas syringae pv. syringae from Belgium. They were identified as P. syringae pv. syringae with a Biolog similarity of 0.57-0.86 and FAMEs similarity of 0.58-0.81. The bacterium was reisolated from the symptomatic plants and blossoms. Identification as P. syringae pv. syringae was confirmed by using PCR primers and sequence tests, and compared with the above-mentioned results. The data supported the fact that the pear blossom and bud blast in China could be caused by P. syringae pv. syringae.

Identification of QTLs Associated with Resistance to Pseudomonas syringae pv.Glycinea in Soybean(Glycine max(L.)Merr)

Soybean bacterial spot disease caused by Pseudomonas syringae pv.Glycinea which is a bacterial disease seriously affects soybean yield.Ten soybean germplasms and recombinant inbred lines(RILs)population were used to identify the resistant trait after inoculated with P.sg(P.sgneau001)in this study.High-density genetic mapping was obtained by specific length amplified fragment sequencing(SLAF-seq)of 149 RILs population which was derived from the crossing between Charleston and Dongnong594.The results indicated that 10 germplasm resources had four resistant germplasms included highly resistant cultivar Charleston,four susceptible varieties included Dongnong594 and two moderately resistant cultivars.Five quantitative trait locus(QTLs)were detected in RILs population by the composite interval mapping(CIM)method,and located on Linkage Group(LG)D1b(chromosome two),LG C2(chromosome six)and LG H(chromosome 12),respectively.LOD scores ranged from 2.68 to 4.95 and the phenotypic variation percentage was from 6%to 11%.Six candidate genes were detected,according to the result of gene annotation information.Four of them had relationship with protein kinase activity,protein phosphorylation and leucine rich repeat(LRR)transmembrane protein,which had high expression after inoculated with P.sg by qRT-PCR.

Role of Coronatine in Inducing Infection and Improving Resistance to Pseudomonas syringae pv.mori in Mulberry Seedlings

Mulberry bacterial blight is caused by Pseudomonas syringae pv. mori. Coronatine （COR）, a phytotoxin and phytohormone produced by several strains of Pseudomonas syringae, is suggested to have an important role in pathogen-plant interaction. The aim of our study was to examine the influence of COR on mul- berry in the process of pathogen infection. Results showed that COR could suppress stomatal closure induced by pathogen-associated molecular patterns （PAMPS）, assist pathogenic bacteria into the leaves, and promote bacterial proliferation in the tissues. High-concentration （1 μmol/L） exogenous COR and COR-producing bacterial pathogen induced chlorosis symptom and decrease of chlorophyll content, contrary to the effects of low-concentration （0.001 μmol/L） exogenous COR and non-COR-preducing bacterial pathogen. Treatments with COR and DC3000 enhanced the production of reactive oxygen species （ ROS）, namely, hydrogen peroxide （H2O2 ） and superexide anion （O2-）, but there were two H2O2 peaks at 1 -3 hpi and 8 -24 hpi in the DC3000-treated leaves while only one peak at 1 -3 h was observed 1 -3 h in the COR-treated ones. H202 could kill the pathogenic bacteria, on the other hand, it also acted as an upstream signaling molecule to promote nitric oxide （NO） production to further participate in the signaling pathway. Enzymatic antioxidant systems （superoxide dismutase, peroxidase and catalase） and defensive enzyme systems （lipoxygenase, polyphenol oxidase and phenylalnine ammonialyase） were activated by COR. Therefore, COR could cooperate on the inva- sion and proliferation of COR-producing bacterial pathogens, and induce the chloresis symptom in mulberry. At the same time, exogenously applied COR also could enhance the resistance to P. syringae pv. mori by production of signal molecules to activate signaling pathway and promoting defense-related metabolism.

Natural Variation in Tomato Reveals Differences in the Recognition of AvrPto and AvrPtoB Effectors from Pseudomonas syringae

The Pto protein kinase from Solanum pimpinellifolium interacts with Pseudomonas syringae effectors AvrPto or AvrPtoB to activate effector-triggered immunity. The previously solved crystal structures of the AvrPto-Pto and AvrPtoB-Pto complexes revealed that Pro binds each effector through both a shared and a unique interface. Hera we use natural variation in wild species of tomato to further investigate Pto recognition of these two effectors. One species, Solanum chmielewskU, was found to have many accessions that recognize only AvrPtoB. The Pto ortholog from one of these accessions was responsible for recognition of AvrPtoB and it differed from Solanum pimpinellifolium Pto by only 14 amino acids, including two in the AvrPto-specific interface, glutamate-49/glycine-51. Converting these two residues to those in Pro （histidine-49/valine-51） did not restore recognition of AvrPto. Subsequent experiments revealed that a single substitution of a histidine-to-aspartate at position 193 in Pto, which is not near the AvrPto- specific interface, was sufficient for conferring recognRion of AvrPto in plant cells. The reciprocal substi- tution of aspartate-to-histidine-193 in Pto abolished AvrPto recognition, confirming the importance of this residue. Our results reveal new aspects about effector recognition by Pto and demonstrate the value of using natural variation to understand the interaction between resistance proteins and pathogen effectors.

Two plant NLR proteins confer strain-specific resistance conditioned by an effector from Pseudomonas syringae pv.actinidiae

Pseudomonas syringae pv.actinidiae(Psa)causes bacterial canker,a devastating disease threatening the Actinidia fruit industry.In a search for non-host resistance genes against Psa,we find that the nucleotidebinding leucine-rich repeat receptor(NLR)protein ZAR1 from both Arabidopsis and Nicotiana benthamiana(Nb)recognizes Hop Z5 and triggers cell death.The recognition requires ZED1 in Arabidopsis and JIM2 in Nb plants,which are members of the ZRK pseudokinases and known components of the ZAR1 resistosome.Surprisingly,Arabidopsis ZAR1 and RPM1,another NLR known to recognize Hop Z5,confer disease resistance to Hop Z5 in a strain-specific manner.Thus,ZAR1,but not RPM1,is solely required for resistance to P.s.maculicola ES4326(Psm)carrying hop Z5,whereas RPM1 is primarily required for resistance to P.s.tomato DC3000(Pst)carrying hop Z5.Furthermore,the ZAR1-mediated resistance to Psm hop Z5 in Arabidopsis is insensitive to SOBER1,which encodes a deacetylase known to suppress the RPM1-mediated resistance to Pst hop Z5.In addition,hop Z5 enhances P.syringae virulence in the absence of ZAR1 or RPM1 and that SOBER1 abolishes such virulence function.Together the study suggests that ZAR1 may be used for improving Psa resistance in Actinidia and uncovers previously unknown complexity of effectortriggered immunity and effector-triggered virulence.

Establishment of a Loop-mediated Isothermal Amplification Method for Rice Bacterial Leaf Brown Spot Disease

Rice bacterial leaf brown spot disease caused by Pseudomonas syringae pv.syringae(Pss)is a major disease on rice.In recent years,Pss has emerged worldwide,seriously affecting rice production.It is very important to establish a rapid detection method of Pss for the diagnosis and prevention of this disease.In order to robust and accurately diagnose the rice bacterial leaf brown spot disease in the field and laboratory,an assay system for the Pss was developed in this study,and the specific sequence of hrcN was used as the target,based on loop-mediated isothermal amplification(LAMP).The best detection system was MgSO 48 mmol·L^(-1),Bst DNA polymerase 8 U,dNTP 1.4 mmol·L^(-1),the ratio of internal and outer primers was 2:1,the reaction temperature was 63℃,the reaction time was 45 min,and the lowest sensitivity was 104 CFU·mL^(-1).This results provided an accurate and robust method for laboratory and field diagnosis of bacterial leaf brown spot disease of rice.

Integrated Green Prevention and Control Techniques for Kiwifruit Canker Disease in "Guichang" Kiwifruit in Xiuwen County, Guizhou Province

Kiwifruit canker disease seriously affects the yield and quality of"Guichang"kiwifruit in Xiuwen County,Guizhou Province.In order to scientifically,safely,greenly and efficiently prevent and control the disease,theory was combined with prevention and control techniques to optimize existing prevention and control techniques,so as to improve the production yield and quality of kiwifruit.Specifically,biocontrol strains targeting local kiwifruit canker disease were screened,and reduced and mixed use of agrochemicals with improved efficiency was studied;and the effects and application techniques of disease resistance inducers and bioorganic fertilizers in inducing systemic disease resistance in kiwifruit trees were explored,and finally,an integrated green prevention and control scheme for kiwifruit canker disease that is suitable for kiwifruit production areas in Guizhou Province and has strong operability was proposed.This study provides technical support for green,efficient,standardized production technical services and sustainable and healthy development of kiwifruit industry.

Construction of Lescpth5 Bait Vector and Detection of Its Self-activated Activity

[ Objective ] The paper was to construct bait vector pGBKTT-Lescpth5, and detect its self-activated transcription activity and the toxicity on yeast cells. [ Method] Lescpth5 was amplified by PCR technique, and connected into bait vector pGBKTT. The recombined vector was transformed into yeast competent ceils AH109 and carried out self-activated detection and toxicity detection in the au.xotrephic medium. [ Result] Digestion and sequencing results showed that bait vector pGBKT7-Lescpth5 was suecessfully constructed with correct reading frame. Self-activated activity and toxicity detection results showed that bait vector had no self- activated activity on yeast strain AH109, which also had no toxicity on yeast. [Condusion] Bait vector successfully constructed could be used in yeast two-hybrid system, which laid the foundation for screening of cDNA library in the next step.

Gc-Ms Analysis and Antibacterial Activity of Mangosteen Leaf Extracts against Plant Pathogenic Bacteria

The potential of Garcinia mangostana as a biological control agent against plant pathogenic bacteria which decrease the quality and volume of crop production worldwide was assessed. Mangosteen leaves were extracted by maceration using chloroform, n-hexane, and methanol. For the in vitro antibacterial activity, two dissimilar species of plant pathogenic bacteria: Pseudomonas syringe pv. tomato and Xanthomonas oryzae pv. oryzae were acquired. Four different concentrations, 12.5, 25, 50, and 100 mg/ml were obtained through the cup-plate agar diffusion technique. Streptomycin sulphate at 30 μg/ml concentration was set as the positive control, whereas every respective solvent used in the leaf extraction was set as the negative control. The results have shown that, only methanol extract demonstrated antibacterial activity when tested on the plant pathogenic bacteria. The highest diameter of inhibition zones was observed in X. oryzae pv. oryzae, at all range of concentrations, followed by P. syringae pv. tomato. The least methanol extract concentration utilised in determination of minimum inhibitory concentration (MIC) assay was at 1.562 mg/ml, inhibiting X. oryzae pv. oryzae, followed by P. syringe pv. tomato at a concentration 3.125 mg/ml. Antibacterial impacts of the most effectual extract of mangosteen crude were supported by the existence of chemical components identified by GC-MS. Cycloartenol, Caryophyllene, Docosane, Phenol, 4,4-Methylenebis (2,6-di-tert-butylphenol) and Chromium were noted as key compounds in the mangosteen leaf extract, which were perhaps causing the antibacterial activity.

猕猴桃溃疡病菌Ⅲ型效应蛋白HopAZ1功能研究与互作蛋白鉴定

细菌Ⅲ型分泌系统可向寄主植物细胞分泌多种效应蛋白,在丁香假单胞菌猕猴桃致病变种(Psa)侵染猕猴桃致病中发挥关键作用。但是,尚不清楚Psa如何利用这些效应蛋白与寄主互作。Psa的5个生物型群体具有不同种类的效应子,但其中共有的14个效应子可能是参与病菌与寄主亲和互作的关键因子。本研究系统调查了其中HopAZ1的功能。结果显示,HopAZ1广泛分布于丁香假单胞菌的不同类群,在Psa不同生物型群体遗传分化前已经存在,且可能由于在致病中具有重要功能而被纯化选择;HopAZ1在Psa侵染猕猴桃早期被诱导表达,敲除突变体对不同猕猴桃枝条的致病力均显著上升;进而通过构建猕猴桃酵母文库和酵母双杂交筛选,发现HopAZ1可能与猕猴桃抗病相关蛋白Cp1(Acc23383)、PR5(Acc28852)互作。这表明HopAZ1是Psa与猕猴桃互作的重要因子,可能通过与Cp1或PR5互作激发一定程度的抗病反应。这为深入解析HopAZ1的作用机制及猕猴桃抗病基因的挖掘提供了重要依据。

MYC2： The Master in Action

Jasmonates （JAs） are plant hormones with essential roles in plant defense and development. The basic- helix-loop-helix （bHLH） transcription factor （TF） MYC2 has recently emerged as a master regulator of most aspects of the jasmonate （JA） signaling pathway in Arabidopsis. MYC2 coordinates JA-mediated defense responses by antagonistically regulating two different branches of the JA signaling pathway that determine resistance to pests and pathogens, respectively. MYC2 is required for induced systemic resistance （ISR） triggered by beneficial soil microbes while MYC2 function is targeted by pathogens during effector-mediated suppression of innate immunity in roots. Another notable function of MYC2 is the regulation of crosstalk between the signaling pathways of JA and those of other phytohormones such as abscisic acid （ABA）, salicylic acid （SA）, gibberellins （GAs）, and auxin （IAA）. MYC2 also regulates interactions between JA signaling and light, phytochrome signaling, and the circadian clock, MYC2 is involved in JA-regulated plant development, lateral and adventitious root formation, flowering time, and shade avoidance syndrome. Related bHLH TFs MYC3 and MYC4 also regulate both overlapping and distinct MYC2-regulated functions in Arabidopsis while MYC2 orthologs act as ＇master switches＇ that regulate JA-mediated biosynthesis of secondary metabolites. Here, we briefly review recent studies that revealed mechanistic new insights into the mode of action of this versatile TF.

Post-Translational Derepression of Invertase Activity in Source Leaves via Down-Regulation of Invertase Inhibitor Expression Is Part of the Plant Defense Response

There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Posttranslational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between invertase and the invertase inhibitor proteins. Upon pathogen infection, the invertase activity is released by repression of invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response.

Circadian Clock-Regulated Phosphate Transporter PHT4;1 Plays an Important Role in Arabidopsis Defense

The Arabidopsis accelerated cell death 6-1 （acd6-1） mutant shows constitutive defense, cell death, and ex- treme dwarf phenotypes. In a screen for acd6-1 suppressors, we identified a mutant that was disrupted by a T-DNA in the PHOSPHATE TRANSPORTER 4;1 （PHT4;1） gene. The suppressor mutant pht4;1-1 is dominant, expresses truncated PHT4;1 transcripts, and is more susceptible to virulent Pseudomonas syringae strains but not to several avirulent strains. Treat- ment with a salicylic acid （SA） agonist induced a similar level of resistance in Col-0 and pht4;1-1, suggesting that PHT4;1 acts upstream of the SA pathway. Genetic analysis further indicates that PHT4,1 contributes to SID2-dependent and -in- dependent pathways. Transgenic expression of the DNA fragment containing the PHT4;1-1 region or the full-length PHT4;1 gene in wild-type conferred enhanced susceptibility to Pseudomonas infection. Interestingly, expression of PHT4;1 is reg- ulated by the circadian clock. Together, these data suggest that the phosphate transporter PHT4;1 is critical for basal defense and also implicate a potential role of the circadian clock in regulating innate immunity of Arabidopsis.

Antagonism of Transcription Factor MYC2 by EDS1/PAD4 Complexes Bolsters Salicylic Acid Defense in Arabidopsis Effector-Triggered Immunity

In plant immunity, pathogen-activated intracellular nucleotide binding/leucine rich repeat （NLR） receptors mobilize disease resistance pathways, but the downstream signaling mechanisms remain obscure. Enhanced disease susceptibility 1 （EDS1） controls transcriptional reprogramming in resistance triggered by Toll-lnterleukinl-Receptor domain （TIR）-family NLRs （TNLs）. Transcriptional induction of the salicylic acid （SA） hormone defense sector provides one crucial barrier against biotrophic pathogens. Here, we present genetic and molecular evidence that in Arabidopsis an EDS1 complex with its partner PAD4 inhibits MYC2, a master regulator of SA-antagonizing jasmonic acid （JA） hormone pathways. In the TNL immune response, EDSl/PAD4 interference with MYC2 boosts the SA defense sector independently of EDS1-induced SA synthesis, thereby effectively blocking actions of a potent bacterial JA mimic, coronatine （COR）. We show that antagonism of MYC2 occurs after COR has been sensed inside the nucleus but before or coincident with MYC2 binding to a target promoter, pANAC019. The stable interaction of PAD4 with MYC2 in planta is competed by EDS1-PAD4 complexes. However, suppression of MYC2-promoted genes requires EDS1 together with PAD4, pointing to an essential EDS1-PAD4 heterodimer activity in MYC2 inhibition. Taken together, these results uncover an immune receptor signaling circuit that intersects with hormone pathway crosstalk to reduce bacterial pathogen growth.

From filaments to function: The role of the plant actin cytoskeleton in pathogen perception,signaling and immunity

The eukaryotic actin cytoskeleton is required for numerous cellular processes, including cell shape, development and movement, gene expression and signal transduction, and response to biotic and abiotic stress. In recent years,research in both plants and animal systems have described a function for actin as the ideal surveillance platform, linking the function and activity of primary physiological processes to the immune system. In this review, we will highlight recent advances that have defined the regulation and breadth of function of the actin cytoskeleton as a network required for defense signaling following pathogen infection. Coupled with an overview of recent work demonstrating specific targeting of the plant actin cytoskeleton by a diversity of pathogens,including bacteria, fungi and viruses, we will highlight the importance of actin as a key signaling hub in plants, one that mediates surveillance of cellular homeostasis and the activation of specific signaling responses following pathogen perception. B4 ased on the studies highlighted herein, we propose a working model that posits changes in actin filament organization is in and of itself a highly specific signal, which induces, regulates and physically directs stimulus-specific signaling processes, most importantly, those associated with response to pathogens.

Regulatory and Functional Aspects of Indolic Metabolism in Plant Systemic Acquired Resistance

Tryptophan-derived, indolic metabolites possess diverse functions in Arabidopsis innate immunity to microbial pathogen infection. Hers, we investigate the functional role and regulatory characteristics of indolic metabolism in Arabidopsis systemic acquired resistance （SAR） triggered by the bacterial pathogen Pseudomonas syringae. Indolic metabolism is broadly activated in both P. syringae-inoculated and distant, non-inoculated leaves. At inoculation sites, camalexin, indol-3-ylmethylamine （13A）, and indole-3-carboxylic acid （ICA） are the major accumulating compounds. Camalexin accumulation is positively affected by MYB122, and the cytochrome P450 genes CYP81F1 and CYP81F2. Local 13A production, by contrast, occurs via indole glucosinolate breakdown by PEN2- dependent and independent pathways. Moreover, exogenous application of the defense hormone salicylic acid stimulates 13A generation at the expense of its precursor indol-3-ylmethylglucosinolate （13M）, and the SAR regulator pipecolic acid primes plants for enhanced P. syringae-induced activation of distinct branches of indolic metabolism. In uninfected systemic tissue, the metabolic response is more specific and associated with enhanced levels of the indolics 13A, ICA, and indole-3-carbaldehyde （ICC）. Systemic indole accumulation fully depends on functional CYP79B2/3, PEN2, and MYB34/51/122, and requires functional SAR signaling. Genetic analyses suggest that systemi- cally elevated indoles are dispensable for SAR and associated systemic increases of salicylic acid. However, soil-grown but not hydroponically -cultivated cyp79b2/3 and pen2 plants, both defective in indolic secondary metabolism, exhibit pre-induced immunity, which abrogates their intrinsic ability to induce SAR.

Robust transcriptional indicators of immune cell death revealed by spatiotemporal transcriptome analyses

Recognition of a pathogen by the plant immune system often triggers a form of regulated cell death traditionally known as the hypersensitive response(HR).This type of cell death occurs precisely at the site of pathogen recognition,and it is restricted to a few cells.Extensive research has shed light on how plant immune receptors are mechanistically activated.However,two central key questions remain largely unresolved:how does cell death zonation take place,and what are the mechanisms that underpin this phenomenon?Consequently,bona fide transcriptional indicators of HR are lacking,which prevents deeper insight into its mechanisms before cell death becomes macroscopic and precludes early or live observation.In this study,to identify the transcriptional indicators of HR we used the paradigmatic Arabidopsis thaliana–Pseudomonas syringae pathosystem and performed a spatiotemporally resolved gene expression analysis that compared infected cells that will undergo HR upon pathogen recognition with bystander cells that will stay alive and activate immunity.Our data revealed unique and time-dependent differences in the repertoire of differentially expressed genes,expression profiles,and biological processes derived from tissue undergoing HR and that of its surroundings.Furthermore,we generated a pipeline based on concatenated pairwise comparisons between time,zone,and treatment that enabled us to define 13 robust transcriptional HR markers.Among these genes,the promoter of an uncharacterized AAA-ATPase was used to obtain a fluorescent reporter transgenic line that displays a strong spatiotemporally resolved signal specifically in cells that will later undergo pathogen-triggered cell death.This valuable set of genes can be used to define cells that are destined to die upon infection with HR-triggering bacteria,opening new avenues for specific and/or high-throughput techniques to study HR processes at a single-cell level.

A versatile Tn7 transposon-based bioluminescence tagging tool for quantitative and spatial detection of bacteria in plants

Investigation of plant-bacteria interactions requires quantification of in planta bacterial titers by means of cumbersome and time-consuming colony-counting assays.Here,we devised a broadly applicable tool for bioluminescence-based quantitative and spatial detection of bacteria in plants.We developed vectors that enable Tn7 transposon-mediated integration of the luxCDABEluciferase operon into a specific genomic location found ubiquitously across bacterial phyla.These vectors allowed for the generation of bioluminescent transformants of various plant pathogenic bacteria from the genera Pseudomonas,Rhizobium(Agrobacterium),and Ralstonia.Direct luminescence measurements of plant tissues inoculated with bioluminescent Pseudomonas syringae pv.tomato DC3000(Pto-lux)reported bacterial titers as accurately as conventional colony-counting assays in Arabidopsis thaliana,Solanum lycopersicum,Nicotiana benthamiana,and Marchantia polymorpha.We further showed the usefulness of our vectors in converting previously generated Pto derivatives to isogenic bioluminescent strains.Importantly,quantitative bioluminescence assays using these Pto-lux strains accurately reported the effects of plant immunity and bacterial effectors on bacterial growth,with a dynamic range of four orders of magnitude.Moreover,macroscopic bioluminescence imaging illuminated the spatial patterns of Pto-lux growth in/on inoculated plant tissues.In conclusion,our vectors offer untapped opportunities to develop bioluminescence-based assays for a variety of plant-bacteria interactions.