Transcriptomic Evidence of a Link between Cell Wall Biogenesis, Pathogenesis, and Vigor in Walnut Root and Trunk Diseases.

Crown gall disease (Agrobacterium tumefaciens), crown/root rot disease (Phytophthora spp.), root lesion disease (Pratylenchus vulnus) and tree vigor are key traits affecting the productivity and quality of walnuts in California. Unchallenged hybrid rootstocks were analyzed by RNA-seq to examine pre-formed factors affecting these traits. Enrichment analysis of the differentially expressed genes revealed that the increased expression of cell wall biogenesis-related genes plays a key role in susceptibility to A. tumefaciens, susceptibility to Phytophthora spp. and increased vigor. Analysis of the predicted subcellular loci of the encoded proteins revealed that many gene products associated with vigor and susceptibility were targeted to the plasma membrane and extracellular space, connecting these traits to sustaining barrier function. We observed that RNA processing and splicing, along with predicted nuclear targeting, were associated with resistance to A. tumefaciens, resistance to Phytophthora spp. and low vigor. Four genes within the J. microcarpa QTL region for resistance to A. tumefaciens and Phytophthora spp. were represented among our transcripts, with two of the genes being differentially expressed in association with resistance to A. tumefaciens and decreased vigor. No differential expression related to Phytophthora spp. or P. vulnus resistance was observed in this region. Additionally, the J. microcarpa haplotype expressed more transcripts associated with resistance to A. tumefaciens, Phytophthora spp. and low vigor, but not P. vulnus, than the J. regia haplotype. We also report unique and shared hormone and defense responses associated with each trait. This research suggests a link between cell wall biogenesis, vigor and critical root diseases of walnut.

Transcriptome and Secretome Analyses of Endophyte Methylobacterium mesophilicum and Pathogen Xylella fastidiosa Interacting Show Nutrient Competition.

Xylella fastidiosa is the causal agent of several plant diseases affecting fruit and nut crops. Methylobacterium mesophilicum strain SR1.6/6 was isolated from Citrus sinensis and shown to promote plant growth by producing phytohormones, providing nutrients, inhibiting X. fastidiosa, and preventing Citrus Variegated Chlorosis. However, the molecular mechanisms involved in the interaction among these microbes are still unclear. The present work aimed to analyze physiological and molecular aspects of M. mesophilicum SR1.6/6 and X. fastidiosa 9a5c in co-culture. The transcriptome and secretome analyses indicated that X. fastidiosa down-regulates cell division and transport genes and up-regulates stress via induction of chaperones and pathogenicity-related genes including, the lipase-esterase LesA, a protease, as well as an oligopeptidase in response to M. mesophilicum competition. On the other hand, M. mesophilicum also down-regulated transport genes, except for iron uptake, which was up-regulated. Secretome analysis identified four proteins in M. mesophilicum exclusively produced in co-culture with X. fastidiosa, among these, three are related to phosphorous uptake. These results suggest that M. mesophilicum inhibits X. fastidiosa growth mainly due to nutrient competition for iron and phosphorous, thus promoting X. fastidiosa starvation, besides producing enzymes that degrade X. fastidiosa cell wall, mainly hydrolases. The understanding of these interactions provides a direction for control and management of the phytopathogen X. fastidiosa, and consequently, helps to improve citrus growth and productivity.

SnRK1 kinase-mediated phosphorylation of transcription factor bZIP39 regulates sorbitol metabolism in apple.

Sorbitol is a major photosynthate produced in leaves and transported through the phloem of apple (Malus domestica) and other tree fruits in Rosaceae. Sorbitol stimulates its own metabolism, but the underlying molecular mechanism remains unknown. Here, we show that sucrose nonfermenting 1 (SNF1)-related protein kinase 1 (SnRK1) is involved in regulating the sorbitol-responsive expression of both SORBITOL DEHYDROGENASE 1 (SDH1) and ALDOSE-6-PHOSPHATE REDUCTASE (A6PR), encoding 2 key enzymes in sorbitol metabolism. SnRK1 expression is increased by feeding of exogenous sorbitol but decreased by sucrose. SnRK1 interacts with and phosphorylates the basic leucine zipper (bZIP) transcription factor bZIP39. bZIP39 binds to the promoters of both SDH1 and A6PR and activates their expression. Overexpression of SnRK1 in 'Royal Gala' apple increases its protein level and activity, upregulating transcript levels of both SDH1 and A6PR without altering the expression of bZIP39. Of all the sugars tested, sorbitol is the only 1 that stimulates SDH1 and A6PR expression, and this stimulation is blocked by RNA interference (RNAi)-induced repression of either SnRK1 or bZIP39. These findings reveal that sorbitol acts as a signal regulating its own metabolism via SnRK1-mediated phosphorylation of bZIP39, which integrates sorbitol signaling into the SnRK1-mediated sugar signaling network to modulate plant carbohydrate metabolism.

Affinity Sedimentation and Magnetic Separation With Plant-Made Immunosorbent Nanoparticles for Therapeutic Protein Purification.

The virus-based immunosorbent nanoparticle is a nascent technology being developed to serve as a simple and efficacious agent in biosensing and therapeutic antibody purification. There has been particular emphasis on the use of plant virions as immunosorbent nanoparticle chassis for their diverse morphologies and accessible, high yield manufacturing via plant cultivation. To date, studies in this area have focused on proof-of-concept immunosorbent functionality in biosensing and purification contexts. Here we consolidate a previously reported pro-vector system into a single Agrobacterium tumefaciens vector to investigate and expand the utility of virus-based immunosorbent nanoparticle technology for therapeutic protein purification. We demonstrate the use of this technology for Fc-fusion protein purification, characterize key nanomaterial properties including binding capacity, stability, reusability, and particle integrity, and present an optimized processing scheme with reduced complexity and increased purity. Furthermore, we present a coupling of virus-based immunosorbent nanoparticles with magnetic particles as a strategy to overcome limitations of the immunosorbent nanoparticle sedimentation-based affinity capture methodology. We report magnetic separation results which exceed the binding capacity reported for current industry standards by an order of magnitude.

Founder transformants of cotton (Gossypium hirsutum L.) obtained through the introduction of DS-Red, Rec, Rep and CRISPR/Cas9 expressing constructs for developing base lines of recombinase mediated gene stacking.

Cotton being the major fiber crop across the world is exposed to numerous biotic and abiotic stresses. Genetic transformation of cotton is vital to meet the world's food, feed and fiber demands. Genetic manipulation by randomly transferring the genes emanate variable gene expression. Targeted gene insertion by latest genome editing tools results in predictable expression of genes at a specified location. Gene stacking technology emerged as an adaptive strategy to combat biotic and abiotic stresses by integrating 2-3 genes simultaneously and at a specific site to avoid variable gene expression at diverse locations. This study explains the development of cotton's founder transformants to be used as a base line for multiple gene stacking projects. We introduced Cre and PhiC31 mediated recombination sites to specify the locus of incoming genes. CRISPR-Cas9 gene was integrated for developing CRISPR based founder lines of cotton. Cas9 gene along with gRNA was integrated to target Rep (replication) region of cotton leaf curl virus. Replication region of virus was specifically targeted to diminish further proliferation and preventing the virus to develop new strains. To successfully develop these primary transformants, a model transformation system has been optimized with the red color visualization (DS-Red). Following red color transformation system, three baselines with recombination specified site (Rec), targeted replication region (Rep) and Cas9 founder lines have been developed. These founder transformants are useful for developing recombinase mediated and CRISPR/Cas9 based originator lines of cotton. Moreover, these transformants will set up a base system for all the recombinase mediated gene stacking projects.

De Novo Arginine Synthesis Is Required for Full Virulence of Xanthomonas arboricola pv. juglandis During Walnut Bacterial Blight Disease.

Walnut blight (WB) disease caused by Xanthomonas arboricola pv. juglandis (Xaj) threatens orchards worldwide. Nitrogen metabolism in this bacterial pathogen is dependent on arginine, a nitrogen-enriched amino acid that can either be synthesized or provided by the plant host. The arginine biosynthetic pathway uses argininosuccinate synthase (argG), associated with increased bacterial virulence. We examined the effects of bacterial arginine and nitrogen metabolism on the plant response during WB by proteomic analysis of the mutant strain Xaj argG-. Phenotypically, the mutant strain produced 42% fewer symptoms and survived in the plant tissue with 2.5-fold reduced growth compared with wild type, while showing itself to be auxotrophic for arginine in vitro. Proteomic analysis of infected tissue enabled the profiling of 676 Xaj proteins and 3,296 walnut proteins using isobaric labeling in a data-dependent acquisition approach. Comparative analysis of differentially expressed proteins revealed distinct plant responses. Xaj wild type (WT) triggered processes of catabolism and oxidative stress in the host under observed disease symptoms, while most of the host biosynthetic processes triggered by Xaj WT were inhibited during Xaj argG- infection. Overall, the Xaj proteins revealed a drastic shift in carbon and energy management induced by disruption of nitrogen metabolism while the top differentially expressed proteins included a Fis transcriptional regulator and a peptidyl-prolyl isomerase. Our results show the critical role of de novo arginine biosynthesis to sustain virulence and minimal growth during WB. This study is timely and critical as copper-based control methods are losing their effectiveness, and new sustainable methods are urgently needed in orchard environments.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Advances in Rootstock Breeding of Nut Trees: Objectives and Strategies.

The production and consumption of nuts are increasing in the world due to strong economic returns and the nutritional value of their products. With the increasing role and importance given to nuts (i.e., walnuts, hazelnut, pistachio, pecan, almond) in a balanced and healthy diet and their benefits to human health, breeding of the nuts species has also been stepped up. Most recent fruit breeding programs have focused on scion genetic improvement. However, the use of locally adapted grafted rootstocks also enhanced the productivity and quality of tree fruit crops. Grafting is an ancient horticultural practice used in nut crops to manipulate scion phenotype and productivity and overcome biotic and abiotic stresses. There are complex rootstock breeding objectives and physiological and molecular aspects of rootstock-scion interactions in nut crops. In this review, we provide an overview of these, considering the mechanisms involved in nutrient and water uptake, regulation of phytohormones, and rootstock influences on the scion molecular processes, including long-distance gene silencing and trans-grafting. Understanding the mechanisms resulting from rootstock × scion × environmental interactions will contribute to developing new rootstocks with resilience in the face of climate change, but also of the multitude of diseases and pests.

A Secreted Chorismate Mutase from Xanthomonas arboricola pv. juglandis Attenuates Virulence and Walnut Blight Symptoms.

Walnut blight is a significant above-ground disease of walnuts caused by Xanthomonas arboricola pv. juglandis (Xaj). The secreted form of chorismate mutase (CM), a key enzyme of the shikimate pathway regulating plant immunity, is highly conserved between plant-associated beta and gamma proteobacteria including phytopathogens belonging to the Xanthomonadaceae family. To define its role in walnut blight disease, a dysfunctional mutant of chorismate mutase was created in a copper resistant strain Xaj417 (XajCM). Infections of immature walnut Juglans regia (Jr) fruit with XajCM were hypervirulent compared with infections with the wildtype Xaj417 strain. The in vitro growth rate, size and cellular morphology were similar between the wild-type and XajCM mutant strains, however the quantification of bacterial cells by dPCR within walnut hull tissues showed a 27% increase in XajCM seven days post-infection. To define the mechanism of hypervirulence, proteome analysis was conducted to compare walnut hull tissues inoculated with the wild type to those inoculated with the XajCM mutant strain. Proteome analysis revealed 3296 Jr proteins (five decreased and ten increased with FDR ≤ 0.05) and 676 Xaj417 proteins (235 increased in XajCM with FDR ≤ 0.05). Interestingly, the most abundant protein in Xaj was a polygalacturonase, while in Jr it was a polygalacturonase inhibitor. These results suggest that this secreted chorismate mutase may be an important virulence suppressor gene that regulates Xaj417 virulence response, allowing for improved bacterial survival in the plant tissues.

A comparative genomic analysis of Xanthomonas arboricola pv. juglandis strains reveal hallmarks of mobile genetic elements in the adaptation and accelerated evolution of virulence.

Xanthomonas arboricola pv. juglandis (Xaj) is the most significant aboveground walnut bacterial pathogen. Disease management uses copper-based pesticides which induce pathogen resistance. We examined the genetic repertoire associated with adaptation and virulence evolution in Xaj. Comparative genomics of 32 Xaj strains reveal the possible acquisition and propagation of virulence factors via insertion sequences (IS). Fine-scale annotation revealed a Tn3 transposon (TnXaj417) encoding copper resistance genes acquired by horizontal gene transfer and associated with adaptation and tolerance to metal-based pesticides commonly used to manage pathogens in orchard ecosystems. Phylogenomic analysis reveals IS involvement in acquisition and diversification of type III effector proteins ranging from two to eight in non-pathogenic strains, 16 to 20 in pathogenic strains, besides six other putative effectors with a reduced identity degree found mostly among pathogenic strains. Yersiniabactin, xopK, xopAI, and antibiotic resistance genes are also located near ISs or inside genomic islands and structures resembling composite transposons.

Co-located quantitative trait loci mediate resistance to Agrobacterium tumefaciens, Phytophthora cinnamomi, and P. pini in Juglans microcarpa × J. regia hybrids.

Soil-borne plant pathogens represent a serious threat that undermines commercial walnut (Juglans regia) production worldwide. Crown gall, caused by Agrobacterium tumefaciens, and Phytophthora root and crown rots, caused by various Phytophthora spp., are among the most devastating walnut soil-borne diseases. A recognized strategy to combat soil-borne diseases is adoption of resistant rootstocks. Here, resistance to A. tumefaciens, P. cinnamomi, and P. pini is mapped in the genome of Juglans microcarpa, a North American wild relative of cultivated walnut. Half-sib J. microcarpa mother trees DJUG 31.01 and DJUG 31.09 were crossed with J. regia cv. Serr, producing 353 and 400 hybrids, respectively. Clonally propagated hybrids were genotyped by sequencing to construct genetic maps for the two populations and challenged with the three pathogens. Resistance to each of the three pathogens was mapped as a major QTL on the long arm of J. microcarpa chromosome 4D and was associated with the same haplotype, designated as haplotype b, raising the possibility that the two mother trees were heterozygous for a single Mendelian gene conferring resistance to all three pathogens. The deployment of this haplotype in rootstock breeding will facilitate breeding of a walnut rootstock resistant to both crown gall and Phytophthora root and crown rots.

Two UGT84A Family Glycosyltransferases Regulate Phenol, Flavonoid, and Tannin Metabolism in Juglans regia (English Walnut).

We showed previously that gallic acid is produced in walnut from 3-dehydroshikimate by a shikimate dehydrogenase (JrSkDH). This study focuses on the next step in the hydrolysable tannin pathway, the formation of 1-O-galloyl-ß-D-glucose from the phenolic gallic acid and UDP glucose by a glycosyltransferase. JrGGT1 (UGT84A73) and JrGGT2 (UGT84A74) are predicted to be two such glycosyltransferases, which we expressed in tobacco plants. GC-MS analysis of the transgenic tobacco revealed moderate, yet significant alterations in plant secondary metabolism, such as depleted phenolic acids, including gallic acid. We postulate that these effects are due to JrGGT1 and JrGGT2 activity, as JrGGT orthologs glycosylate these phenolic compounds in vitro. Moreover, JrGGT expression in tobacco caused upregulation of shikimic acid pathway metabolites and differing responses in phenylpropanoids, such as phenolic acids and flavonoids. In transcriptome analysis of walnut pellicle tissues, both JrGGTs showed substantial and significant expression correlations with the gallic acid-producing JrSkDHs and were highly coexpressed with the genetic circuits constituting the shikimic acid and phenylpropanoid biosynthetic pathways. Verification of JrGGT gene expression by transcriptome analysis of 20 walnut tissues revealed striking similarities with that of the pellicle data, with the greatest expression in roots, wood, buds, and leaves of Juglans regia cv. Chandler: tissues that typically accumulate hydrolysable tannins. Like the transgenic tobacco, pellicle metabolomic analyses revealed that many phenylpropanoids correlated negatively with JrGGT expression, while shikimic acid pathway metabolites correlated positively with JrGGT expression. This research supports the hypothesis that JrGGT1 and JrGGT2 play non-trivial roles in metabolism of phenolic acids, flavonoids, and ostensibly, tannins.

A comparative genomic analysis of Xanthomonas arboricola pv. juglandis strains reveal hallmarks of mobile genetic elements in the adaptation and accelerated evolution of virulence

Xanthomonas arboricola pv. juglandis (Xaj) is the most significant aboveground walnut bacterial pathogen. Disease management uses copper-based pesticides which induce pathogen resistance. We examined the genetic repertoire associated with adaptation and virulence evolution in Xaj. Comparative genomics of 32 Xaj strains reveal the possible acquisition and propagation of virulence factors via insertion sequences (IS). Fine-scale annotation revealed a Tn3 transposon (TnXaj417) encoding copper resistance genes acquired by horizontal gene transfer and associated with adaptation and tolerance to metal-based pesticides commonly used to manage pathogens in orchard ecosystems. Phylogenomic analysis reveals IS involvement in acquisition and diversification of type III effector proteins ranging from two to eight in non-pathogenic strains, 16 to 20 in pathogenic strains, besides six other putative effectors with a reduced identity degree found mostly among pathogenic strains. Yersiniabactin, xopK, xopAI, and antibiotic resistance genes are also located near ISs or inside genomic islands and structures resembling composite transposons.

N-Benzyl-linoleamide, a Constituent of Lepidium meyenii (Maca), Is an Orally Bioavailable Soluble Epoxide Hydrolase Inhibitor That Alleviates Inflammatory Pain.

Lepidium meyenii (maca), a plant indigenous to the Peruvian Andes, recently has been utilized globally for claimed health or recreational benefits. The search for natural products that inhibit soluble epoxide hydrolase (sEH), with therapeutically relevant potencies and concentrations, led to the present study on bioactive amide secondary metabolites found in L. meyenii, the macamides. Based on known and suspected macamides, 19 possible macamides were synthesized and characterized. The majority of these amides displayed excellent inhibitory potency (IC50 ≈ 20-300 nM) toward the recombinant mouse, rat, and human sEH. Quantitative analysis of commercial maca products revealed that certain products contain known macamides (1-5, 8-12) at therapeutically relevant total concentrations (≥3.29 mg/g of root), while the inhibitory potency of L. meyenii extracts directly correlates with the sum of concentration/IC50 ratios of macamides present. Considering both its in vitro efficacy and high abundance in commercial products, N-benzyl-linoleamide (4) was identified as a particularly relevant macamide that can be utilized for in vivo studies. Following oral administration in the rat, compound 4 not only displayed acceptable pharmacokinetic characteristics but effectively reduced lipopolysaccharide-induced inflammatory pain. Inhibition of sEH by macamides provides a plausible biological mechanism of action to account for several beneficial effects previously observed with L. meyenii treatments.

Comparative Proteomic Analysis of Walnut (Juglans regia L.) Pellicle Tissues Reveals the Regulation of Nut Quality Attributes.

Walnuts (Juglans regia L.) are a valuable dietary source of polyphenols and lipids, with increasing worldwide consumption. California is a major producer, with 'Chandler' and 'Tulare' among the cultivars more widely grown. 'Chandler' produces kernels with extra light color at a higher frequency than other cultivars, gaining preference by growers and consumers. Here we performed a deep comparative proteome analysis of kernel pellicle tissue from these two valued genotypes at three harvest maturities, detecting a total of 4937 J. regia proteins. Late and early maturity stages were compared for each cultivar, revealing many developmental responses common or specific for each cultivar. Top protein biomarkers for each developmental stage were also selected based on larger fold-change differences and lower variance among replicates, including proteins for biosynthesis of lipids and phenols, defense-related proteins and desiccation stress-related proteins. Comparison between the genotypes also revealed the common and specific protein repertoires, totaling 321 pellicle proteins with differential abundance at harvest stage. The proteomics data provides clues on antioxidant, secondary, and hormonal metabolism that could be involved in the loss of quality in the pellicles during processing for commercialization.

Genetic Analysis of Walnut (Juglans regia L.) Pellicle Pigment Variation Through a Novel, High-Throughput Phenotyping Platform.

Walnut pellicle color is a key quality attribute that drives consumer preference and walnut sales. For the first time a high-throughput, computer vision-based phenotyping platform using a custom algorithm to quantitatively score each walnut pellicle in L* a* b* color space was deployed at large-scale. This was compared to traditional qualitative scoring by eye and was used to dissect the genetics of pellicle pigmentation. Progeny from both a bi-parental population of 168 trees ('Chandler' × 'Idaho') and a genome-wide association (GWAS) with 528 trees of the UC Davis Walnut Improvement Program were analyzed. Color phenotypes were found to have overlapping regions in the 'Chandler' genetic map on Chr01 suggesting complex genetic control. In the GWAS population, multiple, small effect QTL across Chr01, Chr07, Chr08, Chr09, Chr10, Chr12 and Chr13 were discovered. Marker trait associations were co-localized with QTL mapping on Chr01, Chr10, Chr14, and Chr16. Putative candidate genes controlling walnut pellicle pigmentation were postulated.

Proteome Analysis of Walnut Bacterial Blight Disease.

The interaction between the plant host, walnut (Juglans regia; Jr), and a deadly pathogen (Xanthomonas arboricola pv. juglandis 417; Xaj) can lead to walnut bacterial blight (WB), which depletes walnut productivity by degrading the nut quality. Here, we dissect this pathosystem using tandem mass tag quantitative proteomics. Walnut hull tissues inoculated with Xaj were compared to mock-inoculated tissues, and 3972 proteins were identified, of which 3296 are from Jr and 676 from Xaj. Proteins with differential abundance include oxidoreductases, proteases, and enzymes involved in energy metabolism and amino acid interconversion pathways. Defense responses and plant hormone biosynthesis were also increased. Xaj proteins detected in infected tissues demonstrate its ability to adapt to the host microenvironment, limiting iron availability, coping with copper toxicity, and maintaining energy and intermediary metabolism. Secreted proteases and extracellular secretion apparatus such as type IV pilus for twitching motility and type III secretion effectors indicate putative factors recognized by the host. Taken together, these results suggest intense degradation processes, oxidative stress, and general arrest of the biosynthetic metabolism in infected nuts. Our results provide insights into molecular mechanisms and highlight potential molecular tools for early detection and disease control strategies.

Xylella fastidiosa subsp. pauca Strains Fb7 and 9a5c from Citrus Display Differential Behavior, Secretome, and Plant Virulence.

Xylella fastidiosa colonizes the xylem of various cultivated and native plants worldwide. Citrus production in Brazil has been seriously affected, and major commercial varieties remain susceptible to Citrus Variegated Chlorosis (CVC). Collective cellular behaviors such as biofilm formation influence virulence and insect transmission of X. fastidiosa. The reference strain 9a5c produces a robust biofilm compared to Fb7 that remains mostly planktonic, and both were isolated from symptomatic citrus trees. This work deepens our understanding of these distinct behaviors at the molecular level, by comparing the cellular and secreted proteomes of these two CVC strains. Out of 1017 identified proteins, 128 showed differential abundance between the two strains. Different protein families were represented such as proteases, hemolysin-like proteins, and lipase/esterases, among others. Here we show that the lipase/esterase LesA is among the most abundant secreted proteins of CVC strains as well, and demonstrate its functionality by complementary activity assays. More severe symptoms were observed in Nicotiana tabacum inoculated with strain Fb7 compared to 9a5c. Our results support that systemic symptom development can be accelerated by strains that invest less in biofilm formation and more in plant colonization. This has potential application in modulating the bacterial-plant interaction and reducing disease severity.

Deep Learning Neural Network Prediction Method Improves Proteome Profiling of Vascular Sap of Grapevines during Pierce's Disease Development.

Plant secretome studies highlight the importance of vascular plant defense proteins against pathogens. Studies on Pierce's disease of grapevines caused by the xylem-limited bacterium Xylella fastidiosa (Xf) have detected proteins and pathways associated with its pathobiology. Despite the biological importance of the secreted proteins in the extracellular space to plant survival and development, proteome studies are scarce due to methodological challenges. Prosit, a deep learning neural network prediction method is a powerful tool for improving proteome profiling by data-independent acquisition (DIA). We explored the potential of Prosit's in silico spectral library predictions to improve DIA proteomic analysis of vascular leaf sap from grapevines with Pierce's disease. The combination of DIA and Prosit-predicted libraries increased the total number of identified grapevine proteins from 145 to 360 and Xf proteins from 18 to 90 compared to gas-phase fractionation (GPF) libraries. The new proteins increased the range of molecular weights, assisted in the identification of more exclusive peptides per protein, and increased identification of low-abundance proteins. These improvements allowed identification of new functional pathways associated with cellular responses to oxidative stress, to be investigated further.

Antimicrobial activity of Epsilon-Poly-L-lysine against phytopathogenic bacteria.

Antimicrobial peptides (AMPs) are components of immune defense in many organisms, including plants. They combat pathogens due to their antiviral, antifungal and antibacterial properties, and are considered potential therapeutic agents. An example of AMP is Epsilon-Poly-L-lysine (EPL), a polypeptide formed by ~ 25 lysine residues with known antimicrobial activity against several human microbial pathogens. EPL presents some advantages such as good water solubility, thermal stability, biodegradability, and low toxicity, being a candidate for the control of phytopathogens. Our aim was to evaluate the antimicrobial activity of EPL against four phytobacterial species spanning different classes within the Gram-negative phylum Proteobacteria: Agrobacterium tumefaciens (syn. Rhizobium radiobacter), Ralstonia solanacearum, Xanthomonas citri subsp. citri (X. citri), and Xanthomonas euvesicatoria. The minimum inhibitory concentration (MIC) of the peptide ranged from 80 µg/ml for X. citri to 600 µg/ml for R. solanacearum and X. euvesicatoria. Two hours of MIC exposure led to pathogen death due to cell lysis and was enough for pathogen clearance. The protective and curative effects of EPL were demonstrated on tomato plants inoculated with X. euvesicatoria. Plants showed less disease severity when sprayed with EPL solution, making it a promising natural product for the control of plant diseases caused by diverse Proteobacteria.

High-quality chromosome-scale assembly of the walnut (Juglans regia L.) reference genome.

BACKGROUND: The release of the first reference genome of walnut (Juglans regia L.) enabled many achievements in the characterization of walnut genetic and functional variation. However, it is highly fragmented, preventing the integration of genetic, transcriptomic, and proteomic information to fully elucidate walnut biological processes. FINDINGS: Here, we report the new chromosome-scale assembly of the walnut reference genome (Chandler v2.0) obtained by combining Oxford Nanopore long-read sequencing with chromosome conformation capture (Hi-C) technology. Relative to the previous reference genome, the new assembly features an 84.4-fold increase in N50 size, with the 16 chromosomal pseudomolecules assembled and representing 95% of its total length. Using full-length transcripts from single-molecule real-time sequencing, we predicted 37,554 gene models, with a mean gene length higher than the previous gene annotations. Most of the new protein-coding genes (90%) present both start and stop codons, which represents a significant improvement compared with Chandler v1.0 (only 48%). We then tested the potential impact of the new chromosome-level genome on different areas of walnut research. By studying the proteome changes occurring during male flower development, we observed that the virtual proteome obtained from Chandler v2.0 presents fewer artifacts than the previous reference genome, enabling the identification of a new potential pollen allergen in walnut. Also, the new chromosome-scale genome facilitates in-depth studies of intraspecies genetic diversity by revealing previously undetected autozygous regions in Chandler, likely resulting from inbreeding, and 195 genomic regions highly differentiated between Western and Eastern walnut cultivars. CONCLUSION: Overall, Chandler v2.0 will serve as a valuable resource to better understand and explore walnut biology.