ResearchPad - plant-pathogens https://www.researchpad.co Default RSS Feed en-us © 2020 Newgen KnowledgeWorks <![CDATA[Mycelial biomass estimation and metabolic quotient of <i>Lentinula edodes</i> using species-specific qPCR]]> https://www.researchpad.co/article/elastic_article_15715 Lentinula edodes, commonly known as shiitake, is an edible mushroom that is cultivated and consumed around the globe, especially in Asia. Monitoring mycelial growth inside a woody substrate is difficult, but it is essential for effective management of mushroom cultivation. Mycelial biomass also affects the rate of wood decomposition under natural conditions and must be known to determine the metabolic quotient, an important ecophysiological parameter of fungal growth. Therefore, developing a method to measure it inside a substrate would be very useful. In this study, as the first step in understanding species-specific rates of fungal decomposition of wood, we developed species-specific primers and qPCR procedures for L. edodes. We tested primer specificity using strains of L. edodes from Japan and Southeast Asia, as well as related species of fungi and plant species for cultivation of L. edodes, and generated a calibration curve for quantification of mycelial biomass in wood dust inoculated with L. edodes. The qPCR procedure we developed can specifically detect L. edodes and allowed us to quantify the increase in L. edodes biomass in wood dust substrate and calculate the metabolic quotient based on the mycelial biomass and respiration rate. Development of a species-specific method for biomass quantification will be useful for both estimation of mycelial biomass and determining the kinetics of fungal growth in decomposition processes.

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<![CDATA[<i>Xylella fastidiosa</i> subsp. <i>pauca</i> and olive produced lipids moderate the switch adhesive versus non-adhesive state and <i>viceversa</i>]]> https://www.researchpad.co/article/elastic_article_14717 Global trade and climate change are re-shaping the distribution map of pandemic pathogens. One major emerging concern is Xylella fastidiosa, a tropical bacterium recently introduced into Europe from America. In last decades, X. fastidiosa was detected in several European countries. X. fastidiosa is an insect vector-transmitted bacterial plant pathogen associated with severe diseases in a wide range of hosts. X. fastidiosa through a tight coordination of the adherent biofilm and the planktonic states, invades the host systemically. The planktonic phase is correlated to low cell density and vessel colonization. Increase in cell density triggers a quorum sensing system based on mixture of cis 2-enoic fatty acids—diffusible signalling factors (DSF) that promote stickiness and biofilm. The lipidome profile of Olea europaea L. (cv. Ogliarola salentina) samples, collected in groves located in infected zones and uninfected zones was performed. The untargeted analysis of the lipid profiles of Olive Quick Decline Syndrome (OQDS) positive (+) and negative (-) plants showed a clustering of OQDS+ plants apart from OQDS-. The targeted lipids profile of plants OQDS+ and OQDS- identified a shortlist of 10 lipids that increase their amount in OQDS+ and X. fastidiosa positive olive trees. These lipid entities, provided to X. fastidiosa subsp. pauca pure culture, impact on the dual phase, e.g. planktonic ↔ biofilm. This study provides novel insights on OQDS lipid hallmarks and on molecules that might modulate biofilm phase in X. fastidiosa subsp. pauca.

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<![CDATA[Rediscovering an old foe: Optimised molecular methods for DNA extraction and sequencing applications for fungarium specimens of powdery mildew (Erysiphales)]]> https://www.researchpad.co/article/elastic_article_14476 The purpose of this study was to identify a reliable DNA extraction protocol to use on 25-year-old powdery mildew specimens from the reference collection VPRI in order to produce high quality sequences suitable to address taxonomic phylogenetic questions. We tested 13 extraction protocols and two library preparation kits and found the combination of the E.Z.N.A.® Forensic DNA kit for DNA extraction and the NuGen Ovation® Ultralow System library preparation kit was the most suitable for this purpose.

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<![CDATA[Genome reconstruction of the non-culturable spinach downy mildew <i>Peronospora effusa</i> by metagenome filtering]]> https://www.researchpad.co/article/elastic_article_13800 Peronospora effusa (previously known as P. farinosa f. sp. spinaciae, and here referred to as Pfs) is an obligate biotrophic oomycete that causes downy mildew on spinach (Spinacia oleracea). To combat this destructive many disease resistant cultivars have been bred and used. However, new Pfs races rapidly break the employed resistance genes. To get insight into the gene repertoire of Pfs and identify infection-related genes, the genome of the first reference race, Pfs1, was sequenced, assembled, and annotated. Due to the obligate biotrophic nature of this pathogen, material for DNA isolation can only be collected from infected spinach leaves that, however, also contain many other microorganisms. The obtained sequences can, therefore, be considered a metagenome. To filter and obtain Pfs sequences we utilized the CAT tool to taxonomically annotate ORFs residing on long sequences of a genome pre-assembly. This study is the first to show that CAT filtering performs well on eukaryotic contigs. Based on the taxonomy, determined on multiple ORFs, contaminating long sequences and corresponding reads were removed from the metagenome. Filtered reads were re-assembled to provide a clean and improved Pfs genome sequence of 32.4 Mbp consisting of 8,635 scaffolds. Transcript sequencing of a range of infection time points aided the prediction of a total of 13,277 gene models, including 99 RxLR(-like) effector, and 14 putative Crinkler genes. Comparative analysis identified common features in the predicted secretomes of different obligate biotrophic oomycetes, regardless of their phylogenetic distance. Their secretomes are generally smaller, compared to hemi-biotrophic and necrotrophic oomycete species. We observe a reduction in proteins involved in cell wall degradation, in Nep1-like proteins (NLPs), proteins with PAN/apple domains, and host translocated effectors. The genome of Pfs1 will be instrumental in studying downy mildew virulence and for understanding the molecular adaptations by which new isolates break spinach resistance.

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<![CDATA[Potential role of weather, soil and plant microbial communities in rapid decline of apple trees]]> https://www.researchpad.co/article/5c89776ed5eed0c4847d2c8c

An unusual decline and collapse of young established trees known as “rapid apple decline” (RAD) has become a major concern for apple growers, particularly in the northeastern United States. This decline is characterized by stunted growth, pale yellow to reddish leaves, and tree collapse within weeks after onset of symptoms. We studied declining apple trees to identify potential involvement of abiotic and biotic stresses. We used 16S and ITS to profile bacterial and fungal communities in the soil, rhizosphere, roots, and shoots and tested for the presence of six viruses in scions and rootstocks of symptomatic and asymptomatic trees. The viruses detected were not associated with RAD symptoms. Bacterial and fungal populations were highly variable in plant tissue, soil and rhizosphere samples, with bacteroidetes, firmicutes, proteobacteria, acidobacteria, and actinobacteria the predominant bacterial classes in various samples. ‘Alphaproteobacteria-rickettsiales’, a bacterial class usually reduced in water-limiting soils, had significantly low abundance in root samples of symptomatic trees. Basidiomycota and Ascomycota fungal classes were the most common fungal classes observed, but neither showed differential enrichment between symptomatic and asymptomatic trees. Analyzing weather data showed an extremely cold winter followed by drought in 2015–2016, which likely weakened the trees to make them more susceptible to varied stresses. In addition, similar physical and nutritional soil composition from symptomatic and asymptomatic trees rules out the role of nutritional stress in RAD. Necrotic lesions and wood decay symptoms dispersing from bark or vascular cambium towards the heartwood were observed primarily below the graft union of declining apple trees, suggesting that the rootstock is the originating point of RAD. We speculate that differences in abiotic factors such as moisture levels in declining roots in combination with extreme weather profiles might cause RAD but cannot clearly rule out the involvement of other factors.

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<![CDATA[Evidence of a trans-kingdom plant disease complex between a fungus and plant-parasitic nematodes]]> https://www.researchpad.co/article/5c6dca20d5eed0c48452a801

Disease prediction tools improve management efforts for many plant diseases. Prediction and downstream prevention demand information about disease etiology, which can be complicated for some diseases, like those caused by soilborne microorganisms. Fortunately, the availability of machine learning methods has enabled researchers to elucidate complex relationships between hosts and pathogens without invoking difficult-to-satisfy assumptions. The etiology of a destructive plant disease, Verticillium wilt of mint, caused by the fungus Verticillium dahliae was reevaluated with several supervised machine learning methods. Specifically, the objective of this research was to identify drivers of wilt in commercial mint fields, describe the relationships between these drivers, and predict wilt. Soil samples were collected from commercial mint fields. Wilt foci, V. dahliae, and plant-parasitic nematodes that can exacerbate wilt were quantified. Multiple linear regression, a generalized additive model, random forest, and an artificial neural network were fit to the data, validated with 10-fold cross-validation, and measures of explanatory and predictive performance were compared. All models selected nematodes within the genus Pratylenchus as the most important predictor of wilt. The fungus after which this disease is named, V. dahliae, was the fourth most important predictor of wilt, after crop age and cultivar. All models explained around 50% of the total variation (R2 ≤ 0.46), and exhibited comparable predictive error (RMSE ≤ 1.21). Collectively, these models revealed that the quantitative relationships between two pathogens, mint cultivars and age are required to explain wilt. The ascendance of Pratylenchus spp. in predicting symptoms of a disease assumed to primarily be caused by V. dahliae exposes the underestimated contribution of these nematodes to wilt. This research provides a foundation on which predictive forecasting tools can be developed for mint growers and reminds us of the lessons that can be learned by revisiting assumptions about disease etiology.

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<![CDATA[Viral infection detection using metagenomics technology in six poultry farms of eastern China]]> https://www.researchpad.co/article/5c76fde1d5eed0c484e5afbf

With rapidly increasing animal pathogen surveillance requirements, new technologies are needed for a comprehensive understanding of the roles of pathogens in the occurrence and development of animal diseases. We applied metagenomic technology to avian virus surveillance to study the main viruses infecting six poultry farms in two provinces in eastern China. Cloacal/throat double swabs were collected from 60 birds at each farm according to a random sampling method. The results showed that the method could simultaneously detect major viruses infecting farms, including avian influenza virus, infectious bronchitis virus, Newcastle disease virus, rotavirus G, duck hepatitis B virus, and avian leukemia virus subgroup J in several farms. The test results were consistent with the results from traditional polymerase chain reaction (PCR) or reverse transcription-PCR analyses. Five H9N2 and one H3N8 avian influenza viruses were detected at the farms and were identified as low pathogenic avian influenza viruses according to HA cleavage sites analysis. One detected Newcastle disease virus was classified as Class II genotype I and avirulent type according to F0 cleavage sites analysis. Three avian infectious bronchitis viruses were identified as 4/91, CK/CH/LSC/99I and TC07-2 genotypes by phylogenetic analysis of S1 genes. The viral infection surveillance method using metagenomics technology enables the monitoring of multiple viral infections, which allows the detection of main infectious viruses.

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<![CDATA[Genome-enhanced detection and identification of fungal pathogens responsible for pine and poplar rust diseases]]> https://www.researchpad.co/article/5c648cebd5eed0c484c81ab7

Biosurveillance is a proactive approach that may help to limit the spread of invasive fungal pathogens of trees, such as rust fungi which have caused some of the world’s most damaging diseases of pines and poplars. Most of these fungi have a complex life cycle, with up to five spore stages, which is completed on two different hosts. They have a biotrophic lifestyle and may be propagated by asymptomatic plant material, complicating their detection and identification. A bioinformatics approach, based on whole genome comparison, was used to identify genome regions that are unique to the white pine blister rust fungus, Cronartium ribicola, the poplar leaf rust fungi Melampsora medusae and Melampsora larici-populina or to members of either the Cronartium and Melampsora genera. Species- and genus-specific real-time PCR assays, targeting these unique regions, were designed with the aim of detecting each of these five taxonomic groups. In total, twelve assays were developed and tested over a wide range of samples, including different spore types, different infected plant parts on the pycnio-aecial or uredinio-telial host, and captured insect vectors. One hundred percent detection accuracy was achieved for the three targeted species and two genera with either a single assay or a combination of two assays. This proof of concept experiment on pine and poplar leaf rust fungi demonstrates that the genome-enhanced detection and identification approach can be translated into effective real-time PCR assays to monitor tree fungal pathogens.

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<![CDATA[Blast resistance in Indian rice landraces: Genetic dissection by gene specific markers]]> https://www.researchpad.co/article/5c5217bdd5eed0c4847944c5

Understanding of genetic diversity is important to explore existing gene in any crop breeding program. Most of the diversity preserved in the landraces which are well–known reservoirs of important traits for biotic and abiotic stresses. In the present study, the genetic diversity at twenty-four most significant blast resistance gene loci using twenty-eight gene specific markers were investigated in landraces originated from nine diverse rice ecologies of India. Based on phenotypic evaluation, landraces were classified into three distinct groups: highly resistant (21), moderately resistant (70) and susceptible (70). The landraces harbour a range of five to nineteen genes representing blast resistance allele with the frequency varied from 4.96% to 100%. The cluster analysis grouped entire 161 landraces into two major groups. Population structure along with other parameters was also analyzed to understand the evolution of blast resistance gene in rice. The population structure analysis and principal coordinate analysis classified the landraces into two sub–populations. Analysis of molecular variance showed maximum (93%) diversity within the population and least (7%) between populations. Five markers viz; K3957, Pikh, Pi2–i, RM212and RM302 were strongly associated with blast disease with the phenotypic variance of 1.4% to 7.6%. These resistant landraces will serve as a valuable genetic resource for future genomic studies, host–pathogen interaction, identification of novel R genes and rice improvement strategies.

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<![CDATA[Atlantic West Ophiothrix spp. in the scope of integrative taxonomy: Confirming the existence of Ophiothrix trindadensis Tommasi, 1970]]> https://www.researchpad.co/article/5c52183fd5eed0c4847978e8

We re-describe and confirm the validity of Ophiothrix trindadensis Tommasi, 1970 (Echinodermata: Ophiuroidea). This is a native species from Brazil, however it lacked a type series deposited in scientific collections. The recognition of O. trindadensis was made possible using integrative taxonomy applied to many specimens from the type locality (Trindade Island) as well as from different locations along the Brazilian coast (Araçá Bay and Estuarine Complex of Paranaguá). Initially, 835 specimens were studied and divided into four candidate species (CS) inferred from external morphological characters. Afterwards, the CSs were compared using integrative taxonomy based on external morphology, arm microstructures morphology (arm ossicle), morphometry, and molecular studies (fragments of the mitochondrial genes 16S and COI). Analyses indicated CS1 and CS2 as O. trindadensis, and CS3 as O. angulata, both valid species. CS4 remains O. cf. angulata as more data, including their ecology and physiology, are needed to be definitively clarified. Our integrative investigation using specimens from the type locality overcame the lack of type specimens and increased the reliable identification of O. trindadensis and O. angulata.

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<![CDATA[Comparison of the response of alternative oxidase and uncoupling proteins to bacterial elicitor induced oxidative burst]]> https://www.researchpad.co/article/5c40f80fd5eed0c484386f5b

Plant UCPs are proved to take part in the fine-tuning of mitochondrial ROS generation. It has emerged that mitochondrion can be an important early source of intracellular ROS during plant-pathogen interaction thus plant UCPs must also play key role in this redox fine-tuning during the early phase of plant–pathogen interaction. On the contrary of this well-established assumption, the expression of plant UCPs and their activity has not been investigated in elicitor induced oxidative burst. Thus, the level of plant UCPs both at RNA and protein level and their activity was investigated and compared to AOX as a reference in Arabidopsis thaliana cells due to bacterial harpin treatments. Similar to the expression and activity of AOX, the transcript level of UCP4, UCP5 and the UCP activity increased due to harpin treatment and the consequential oxidative burst. The expression of UCP4 and UCP5 elevated 15-18-fold after 1 h of treatment, then the activity of UCP reached its maximal value at 4h of treatment. The quite rapid activation of UCP due to harpin treatment gives another possibility to fine tune the redox balance of plant cell, furthermore explains the earlier observed rapid decrease of mitochondrial membrane potential and consequent decrease of ATP synthesis after harpin treatment.

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<![CDATA[Choroidal structural analysis in eyes with diabetic retinopathy and diabetic macular edema—A novel OCT based imaging biomarker]]> https://www.researchpad.co/article/5c1966e9d5eed0c484b534e7

Purpose

To evaluate structural changes in the choroid among patients with diabetic macular edema (DME), with varying grades of diabetic retinopathy (DR), using enhance depth imaging spectral domain optical coherence tomography (EDI SD-OCT) scans.

Methods

A cross-sectional study was conducted on 82 eyes with DR and DME and 86 healthy control eyes. Eyes with DME were classified according to the severity of DR as per the international DR severity scale. Sub foveal choroidal thickness (SFCT)was obtained using EDI SD-OCT scans. These scans were binarized into luminal and stromal areas, to derive the choroidal vascularity index (CVI). CVI and SFCT were analyzed between the study and control group using paired-T test. Tukey’s test was used to correlate the differences in CVI and SFCT between different grades of DR. Further analysis was done to look for the effect of DR severity and type of DME on CVI as well as SFCT using correlation coefficient and linear regression analysis.

Results

SFCT was significantly increased in eyes with DME as compared to the controls (334.47±51.81μm vs 284.53±56.45μm, p<0.001), and showed an ascending trend with worsening of DR, though this difference was not statistically significant [mild non-proliferative diabetic retinopathy (NPDR) = 304.33±40.39μm, moderate NPDR = 327.81±47.39μm, severe NPDR = 357.72±62.65μm, proliferative DR (PDR) = 334.59±47.4μm, p-0.09]. CVI was significantly decreased in DME with DR eyes as compared to controls (63.89±1.89 vs 67.51±2.86, p<0.001). CVI was also significantly decreased with worsening DR (mild NPDR = 66.38±0.3, moderate NPDR = 65.28±0.37, severe NPDR = 63.50±0.47, PDR = 61.27±0.9, p<0.001).

Conclusion

SFCT and CVI are dynamic parameters that are affected by DME. Unlike CVI, SFCT is also affected by ocular and systemic factors like edema and hypertension. CVI may be a more accurate surrogate marker for DME and DR and can potentially be used to monitor the progression of DR.

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<![CDATA[Identification of barley powdery mildew resistances in gene bank accessions and the use of gene diversity for verifying seed purity and authenticity]]> https://www.researchpad.co/article/5c141ec3d5eed0c484d2821a

Human activities including those in crop gene banks are subject to errors, especially during seed multiplication and maintenance of seed germination. Therefore, the most serious problem of gene banks is authenticity of the accessions and their genotypic purity. There are many methods for determining the identity of varieties, but comparisons between current data and past records are not easy since the latter are often missing. Breeding barley resistant to powdery mildew caused by Blumeria graminis f. sp. hordei (Bgh) was traditionally based on incorporating major genes into new varieties and the results have been published. Our goal was to identify resistance genes to powdery mildew in accessions of the Czech spring barley core collection and compare these data with earlier information to establish the authenticity of the accessions. Two hundred and twenty-three accessions of the collection including 665 single plant progenies were tested. Sixty-four selected reference isolates of Bgh representing the world diversity of the pathogen were used for resistance tests. Twenty-two known resistance genes were postulated either separately or in combinations. In the collection, 151 homogeneous accessions were found, but the resistances of nine of them were inconsistent with published data and in 12 accessions their authenticity is doubtful. The remaining 72 accessions were heterogeneous and comprised 176 resistance genotypes, 54 of which were probably mechanical admixtures of other varieties. There are several pathogens of cereals, e.g. rusts and mildews, against which many resistance genes in host crops have also been exploited. Knowledge of these resistances can assist in maintaining pure and genuine stocks in gene banks. Seed purity and the authenticity of accessions can subsequently be checked with more advanced methods.

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<![CDATA[Sugarcane apoplast fluid modulates the global transcriptional profile of the diazotrophic bacteria Paraburkholderia tropica strain Ppe8]]> https://www.researchpad.co/article/5c1d5b7ed5eed0c4846ebc6d

The stalk apoplast fluid of sugarcane contains different sugars, organic acids and amino acids that may supply the demand for carbohydrates by endophytic bacteria including diazotrophs P. tropica (syn. B. tropica) strain Ppe8, isolated from sugarcane, is part of the bacterial consortium recommended as inoculant to sugarcane. However, little information has been accumulated regarding this plant-bacterium interaction considering that it colonizes internal sugarcane tissues. Here, we made use of the RNA-Seq transcriptomic analysis to study the influence of sugarcane stalk apoplast fluid on Ppe8 gene expression. The bacterium was grown in JMV liquid medium (100 ml), divided equally and then supplemented with 50 ml of fresh JMV medium or 50 ml of apoplast fluid extracted from sugarcane variety RB867515. Total RNA was extracted 2 hours later, the rRNAs were depleted and mRNAs used to construct libraries to sequence the fragments using Ion Torrent technology. The mapping and statistical analysis were carried out with CLC Genomics Workbench software. The RNA-seq data was validated by RT-qPCR using the reference genes fliP1, paaF, and groL. The data analysis showed that 544 genes were repressed and 153 genes were induced in the presence of apoplast fluid. Genes that induce plant defense responses, genes related to chemotaxis and movements were repressed in the presence of apoplast fluid, indicating that strain Ppe8 recognizes the apoplast fluid as a plant component. The expression of genes involved in bacterial metabolism was regulated (up and down), suggesting that the metabolism of strain Ppe8 is modulated by the apoplast fluid. These results suggest that Ppe8 alters its gene expression pattern in the presence of apoplast fluid mainly in order to use compounds present in the fluid as well as to avoid the induction of plant defense mechanisms. This is a pioneer study showing the role played by the sugarcane apoplast fluid on the global modulation of genes in P. tropica strain Ppe8.

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<![CDATA[Transcriptome-wide responses of adult melon thrips (Thrips palmi) associated with capsicum chlorosis virus infection]]> https://www.researchpad.co/article/5c141e64d5eed0c484d26693

Thrips palmi is a widely distributed major agricultural pest in the tropics and subtropics, causing significant losses in cucurbit and solanaceous crops through feeding damage and transmission of tospoviruses. Thrips palmi is a vector of capsicum chlorosis virus (CaCV) in Australia. The present understanding of transmission biology and potential effects of CaCV on T. palmi is limited. To gain insights into molecular responses to CaCV infection, we performed RNA-Seq to identify thrips transcripts that are differentially-abundant during virus infection of adults. De-novo assembly of the transcriptome generated from whole bodies of T. palmi adults generated 166,445 contigs, of which ~24% contained a predicted open reading frame. We identified 1,389 differentially-expressed (DE) transcripts, with comparable numbers up- (708) and down-regulated (681) in virus-exposed thrips compared to non-exposed thrips. Approximately 59% of these DE transcripts had significant matches to NCBI non-redundant proteins (Blastx) and Blast2GO identified provisional functional categories among the up-regulated transcripts in virus-exposed thrips including innate immune response-related genes, salivary gland and/or gut-associated genes and vitellogenin genes. The majority of the immune-related proteins are known to serve functions in lysosome activity and melanisation in insects. Most of the up-regulated oral and extra-oral digestion-associated genes appear to be involved in digestion of proteins, lipids and plant cell wall components which may indirectly enhance the likelihood or frequency of virus transmission or may be involved in the regulation of host defence responses. Most of the down-regulated transcripts fell into the gene ontology functional category of ‘structural constituent of cuticle’. Comparison to DE genes responsive to tomato spotted wilt virus in Frankliniella occidentalis indicates conservation of some thrips molecular responses to infection by different tospoviruses. This study assembled the first transcriptome in the genus Thrips and provides important data to broaden our understanding of networks of molecular interactions between thrips and tospoviruses.

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<![CDATA[Proteasome-associated HECT-type ubiquitin ligase activity is required for plant immunity]]> https://www.researchpad.co/article/5bfdb41ed5eed0c4845cecbb

Regulated degradation of proteins by the 26S proteasome plays important roles in maintenance and signalling in eukaryotic cells. Proteins are marked for degradation by the action of E3 ligases that site-specifically modify their substrates by adding chains of ubiquitin. Innate immune signalling in plants is deeply reliant on the ubiquitin-26S proteasome system. While progress has been made in understanding substrate ubiquitination during plant immunity, how these substrates are processed upon arrival at the proteasome remains unclear. Here we show that specific members of the HECT domain-containing family of ubiquitin protein ligases (UPL) play important roles in proteasomal substrate processing during plant immunity. Mutations in UPL1, UPL3 and UPL5 significantly diminished immune responses activated by the immune hormone salicylic acid (SA). In depth analyses of upl3 mutants indicated that these plants were impaired in reprogramming of nearly the entire SA-induced transcriptome and failed to establish immunity against a hemi-biotrophic pathogen. UPL3 was found to physically interact with the regulatory particle of the proteasome and with other ubiquitin-26S proteasome pathway components. In agreement, we demonstrate that UPL3 enabled proteasomes to form polyubiquitin chains, thereby regulating total cellular polyubiquitination levels. Taken together, our findings suggest that proteasome-associated ubiquitin ligase activity of UPL3 promotes proteasomal processivity and is indispensable for development of plant immunity.

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<![CDATA[Functional characterization of unique enzymes in Xanthomonas euvesicatoria related to degradation of arabinofurano-oligosaccharides on hydroxyproline-rich glycoproteins]]> https://www.researchpad.co/article/5c0c04edd5eed0c48481d005

In this study, we clarified the functions of three uncharacterized enzymes, XCV2724, XCV2728, and XCV2729, in Xanthomonas euvesicatoria, the causal agent of bacterial spot of tomato and pepper. The genes corresponding to the three enzymes are homologs of hypBA1, hypBA2, and hypAA from Bifidobacterium longum and are unique to Xanthomonas spp. among plant pathogenic bacteria. Functional characterization of the recombinant enzymes expressed using microbial systems revealed that they degrade the arabinofurano-oligosaccharides present on hydroxyproline (Hyp)-rich glycoproteins (HRGPs) such as extensin and solanaceous lectins in plant cell walls. These enzymes work coordinately to degrade the oligosaccharides. First, XeHypAA (XCV2728), belonging to the glycoside hydrolase (GH) 43 family, releases L-arabinose from L-arabinofuranose (Araf)-α1,3-Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara4-Hyp), cleaving its α1,3 bond; second, XeHypBA2 (XCV2729), belonging to the GH121 family, releases the disaccharide Araf-ß1,2-Araf from Araf-ß1,2-Araf-ß1,2-Araf-ß-Hyp (Ara3-Hyp); finally, XeHypBA1 (XCV2724), belonging to GH family 127, releases L-arabinose from Araf-ß-Hyp (Ara-Hyp). In summary, the main oligosaccharide structure of Ara4-Hyp on the HRGPs is degraded to Ara3-Hyp, then to Ara-Hyp, and finally to Ara monosaccharides by the action of these three enzymes. HRGPs containing oligosaccharide substrates have been reported to contribute to plant defense, and interestingly, the promoter region of the operon (xehypBA2 and xehypAA) contains the plant-inducible promoter box for binding the regulator protein HrpX involved in pathogenicity. We then analyzed the expression level of the operon gene in hrp-inducing medium and in plants and constructed gene-deletion mutants. However, although the operon was evidently upregulated by HrpX, three single-gene deletion mutants (ΔxehypBA1, ΔxehypBA2, ΔxehypAA) and even a triple-gene deletion mutant (ΔxehypBA1-BA2-AA) remained pathogenic, and had no effect on nonhost resistance, either, indicating that these three enzymes are not involved in either pathogenicity or nonhost resistance reactions. This is the first report of enzymes in plant pathogenic bacteria that catalyze the degradation of Hyp-linked-L-arabinofuranosides in plant cell walls.

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<![CDATA[Functional analysis of African Xanthomonas oryzae pv. oryzae TALomes reveals a new susceptibility gene in bacterial leaf blight of rice]]> https://www.researchpad.co/article/5b28b5b4463d7e1340e24738

Most Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9–16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1, validating OsERF#123 as a new, bacterial blight S gene.

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<![CDATA[Variation in Virus Symptom Development and Root Architecture Attributes at the Onset of Storage Root Initiation in ‘Beauregard’ Sweetpotato Plants Grown with or without Nitrogen]]> https://www.researchpad.co/article/5989d9fdab0ee8fa60b72d72

It has been shown that virus infections, often symptomless, significantly limit sweetpotato productivity, especially in regions characterized by low input agricultural systems. In sweetpotatoes, the successful emergence and development of lateral roots (LRs), the main determinant of root architecture, determines the competency of adventitious roots to undergo storage root initiation. This study aimed to investigate the effect of some plant viruses on root architecture attributes during the onset of storage root initiation in ‘Beauregard’ sweetpotatoes that were grown with or without the presence of nitrogen. In two replicate experiments, virus-tested plants consistently failed to show visible symptoms at 20 days regardless of nitrogen treatment. In both experiments, the severity of symptom development among infected plants ranged from 25 to 118% when compared to the controls (virus tested plants grown in the presence of nitrogen). The presence of a complex of viruses (Sweet potato feathery mottle virus, Sweet potato virus G, Sweet potato virus C, and Sweet potato virus 2) was associated with 51% reduction in adventitious root number among plants grown without nitrogen. The effect of virus treatments on first order LR development depended on the presence or absence of nitrogen. In the presence of nitrogen, only plants infected with Sweet potato chlorotic stunt virus showed reductions in first order LR length, number, and density, which were decreased by 33%, 12%, and 11%, respectively, when compared to the controls. In the absence of nitrogen, virus tested and infected plants manifested significant reductions for all first order LR attributes. These results provide evidence that virus infection directly influences sweetpotato yield potential by reducing both the number of adventitious roots and LR development. These findings provide a framework for understanding how virus infection reduces sweetpotato yield and could lead to the development of novel strategies to mitigate virus effects on sweetpotato productivity.

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<![CDATA[TAL Effectors Target the C-Terminal Domain of RNA Polymerase II (CTD) by Inhibiting the Prolyl-Isomerase Activity of a CTD-Associated Cyclophilin]]> https://www.researchpad.co/article/5989dacaab0ee8fa60bb3c27

Transcriptional activator-like (TAL) effectors of plant pathogenic bacteria function as transcription factors in plant cells. However, how TAL effectors control transcription in the host is presently unknown. Previously, we showed that TAL effectors of the citrus canker pathogen Xanthomonas citri, named PthAs, targeted the citrus protein complex comprising the thioredoxin CsTdx, ubiquitin-conjugating enzymes CsUev/Ubc13 and cyclophilin CsCyp. Here we show that CsCyp complements the function of Cpr1 and Ess1, two yeast cyclophilins that regulate transcription by the isomerization of proline residues of the regulatory C-terminal domain (CTD) of RNA polymerase II. We also demonstrate that CsCyp, CsTdx, CsUev and four PthA variants interact with the citrus CTD and that CsCyp co-immunoprecipitate with the CTD in citrus cell extracts and with PthA2 transiently expressed in sweet orange epicotyls. The interactions of CsCyp with the CTD and PthA2 were inhibited by cyclosporin A (CsA), a cyclophilin inhibitor. Moreover, we present evidence that PthA2 inhibits the peptidyl-prolyl cis-trans isomerase (PPIase) activity of CsCyp in a similar fashion as CsA, and that silencing of CsCyp, as well as treatments with CsA, enhance canker lesions in X. citri-infected leaves. Given that CsCyp appears to function as a negative regulator of cell growth and that Ess1 negatively regulates transcription elongation in yeast, we propose that PthAs activate host transcription by inhibiting the PPIase activity of CsCyp on the CTD.

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