Botrytis cinerea causes different plant responses in grape (Vitis vinifera) berries during noble and grey rot: diverse metabolism versus simple defence.

The complexity of the interaction between the necrotrophic pathogen Botrytis cinerea and grape berries (Vitis vinifera spp.) can result in the formation of either the preferred noble rot (NR) or the loss-making grey rot (GR), depending on the prevailing climatic conditions. In this study, we focus on the functional gene set of V. vinifera by performing multidimensional scaling followed by differential expression and enrichment analyses. The aim of this study is to identify the differences in gene expression between grape berries in the phases of grey rot, noble rot, and developing rot (DR, in its early stages) phases. The grapevine transcriptome at the NR phase was found to exhibit significant differences from that at the DR and GR stages, which displayed strong similarities. Similarly, several plant defence-related pathways, including plant-pathogen interactions as hypersensitive plant responses were found to be enriched. The results of the analyses identified a potential plant stress response pathway (SGT1 activated hypersensitive response) that was found to be upregulated in the GR berry but downregulated in the NR berry. The study revealed a decrease in defence-related in V. vinifera genes during the NR stages, with a high degree of variability in functions, particularly in enriched pathways. This indicates that the plant is not actively defending itself against Botrytis cinerea, which is otherwise present on its surface with high biomass. This discrepancy underscores the notion that during the NR phase, the grapevine and the pathogenic fungi interact in a state of equilibrium. Conversely the initial stages of botrytis infection manifest as a virulent fungus-plant interaction, irrespective of whether the outcome is grey or noble rot.

Comprehensive analyses of the occurrence of a fungicide resistance marker and the genetic structure in Erysiphe necator populations.

Genetically distinct groups of Erysiphe necator, the fungus causing grapevine powdery mildew infect grapevine in Europe, yet the processes sustaining stable genetic differences between those groups are less understood. Genotyping of over 2000 field samples from six wine regions in Hungary collected between 2017 and 2019 was conducted to reveal E. necator genotypes and their possible differentiation. The demethylase inhibitor (DMI) fungicide resistance marker A495T was detected in all wine regions, in 16% of the samples. Its occurrence differed significantly among wine regions and grape cultivars, and sampling years, but it did not differ between DMI-treated and untreated fields. Multilocus sequence analyses of field samples and 59 in vitro maintained isolates revealed significant genetic differences among populations from distinct wine regions. We identified 14 E. necator genotypes, of which eight were previously unknown. In contrast to the previous concept of A and B groups, European E. necator populations should be considered genetically more complex. Isolation by geographic distance, growing season, and host variety influence the genetic structuring of E. necator, which should be considered both during diagnoses and when effective treatments are planned.

Disentangling the effects of terroir, season, and vintage on the grapevine fungal pathobiome.

The composition, diversity and dynamics of microbial communities associated with grapevines may be influenced by various environmental factors, including terroir, vintage, and season. Among these factors, terroir stands out as a unique possible determinant of the pathobiome, the community of plant-associated pathogens. This study employed high-throughput molecular techniques, including metabarcoding and network analysis, to investigate the compositional dynamics of grapevine fungal pathobiome across three microhabitats (soil, woody tissue, and bark) using the Furmint cultivar. Samples were collected during late winter and late summer in 2020 and 2021, across three distinct terroirs in Hungary's Tokaj wine region. Of the 123 plant pathogenic genera found, Diplodia, Phaeomoniella, and Fusarium displayed the highest richness in bark, wood, and soil, respectively. Both richness and abundance exhibited significant disparities across microhabitats, with plant pathogenic fungi known to cause grapevine trunk diseases (GTDs) demonstrating highest richness and abundance in wood and bark samples, and non-GTD pathogens prevailed soil. Abundance and richness, however, followed distinct patterns Terroir accounted for a substantial portion of the variance in fungal community composition, ranging from 14.46 to 24.67%. Season and vintage also contributed to the variation, explaining 1.84 to 2.98% and 3.67 to 6.39% of the variance, respectively. Notably, significant compositional differences in fungi between healthy and diseased grapevines were only identified in wood and bark samples. Cooccurrence networks analysis, using both unweighted and weighted metrics, revealed intricate relationships among pathogenic fungal genera. This involved mostly positive associations, potentially suggesting synergism, and a few negative relationships, potentially suggesting antagonistic interactions. In essence, the observed differences among terroirs may stem from environmental filtering due to varied edaphic and mesoclimatic conditions. Temporal weather and vine management practices could explain seasonal and vintage fungal dynamics. This study provides insights into the compositional dynamics of grapevine fungal pathobiome across different microhabitats, terroirs, seasons, and health statuses. The findings emphasize the importance of considering network-based approaches in studying microbial communities and have implications for developing improved viticultural plant health strategies.

Botrytis cinerea expression profile and metabolism differs between noble and grey rot of grapes.

Botrytis cinerea, the pathogen causing grey rot (GR) with important economic losses in fruit crops, can also cause noble rot (NR) of grape berries under certain environmental conditions, leading to metabolic and physical changes necessary for producing highly regarded botrytized wines. The functional genes involved in biochemical processes in these harmful vs. beneficial berry rot types are still scarcely understood. We generated and analyzed transcriptomic data from healthy (H), NR and GR grape berries collected in the Tokaj wine region in Hungary. Our study shows that B. cinerea is most active in NR, followed by GR and H berries. In addition, expression profiles differed qualitatively between NR and GR, and to a smaller extent between months. Several functional genes expressed during NR were linked to well-known physico-chemical changes in botrytized grape berries, including berry skin degradation and the formation of metabolites favorable for botrytized wine production. In addition, we found that B. cinerea appeared to express genes involved in the biosynthesis of antimicrobials during NR, but not in GR, which likely contributes to the dominance of this fungus during NR.

Paraffin oil induces resistance against powdery mildew in grapevine through salicylic acid signaling.

BACKGROUND: Mineral oils have been widely used in the pest control of several crops. However, their mode of action is poorly understood, especially in the case of their antifungal properties. The possible direct fungicidal activity and the stress-inducing capability of paraffin oil on grapevine were examined using Vitis vinifera L. cv 'Kékfrankos' cuttings and the fungus Erysiphe necator, the causal agent of powdery mildew. RESULTS: Our experiments demonstrated that paraffin oil does not have fungicide activity on E. necator, but induces significant stress-related changes in grapevine physiology. Elevated H2 O2 production and the accumulation of the phytohormone salicylic acid were measured. Secondary thickening of the cell wall by lignin deposition and the accumulation of phenolic compounds were also observed. Some enzyme activities related to the detoxification of reactive oxygen species, disease response, and the synthesis of lignin were changed in accordance with the determined changes in cell wall composition and H2 O2 levels. CONCLUSION: The results suggest that paraffin oil induces stress responses on grapevine leaves through oxidative burst, and this response is systemized by salicylic acid. The accumulation of lignin and water-soluble phenolics may be directly responsible for the paraffin oil-induced resistance of grapevine against powdery mildew. © 2021 Society of Chemical Industry.

Sexual recombination in the Botrytis cinerea populations in Hungarian vineyards.

Botrytis cinerea (anamorph of Botryotinia fuckeliana) causes gray mold on a high number of crop plants including grapes. In this study, we investigated the genetic properties of a grape pathogenic population of B. cinerea in the area of Eger, Hungary. A total of 109 isolates from 12 areas were sampled. Based on the sequence of the beta-tubulin (tub1) locus, they all belong to group II, a phylogenetic species within B. cinerea. Seventy-four isolates were classified as transposa, with both the Flipper and Boty transposons, and 10 were classified as vacuma, lacking both transposons. The remaining isolates contained either only Flipper (13) or Boty (12). Multilocus analysis of sequences from tub1 and two other loci (elongation factor 1-alpha, tef1, and a minisatellite from the intron of an ATPase, MSB1) led to poor phylogenetic resolution of strains in individual clades. Analysis of five microsatellites (Bc2, Bc3, Bc5, Bc6, and Bc10) resulted in 55 microsatellite haplotypes within the 109 strains. No correlation was detected among individual haplotypes and the presence/absence of Flipper and/or Boty, the geographic origin, or the year of isolation. Application of the index of association, the chi-square test, and the phi test consistently indicated that the population of Hungarian isolates of B. cinerea undergoes sexual reproduction. However, the index of association test suggested the presence of some clonality, and the fixation index showed a low or occasionally moderate level of fixation in the Flipper populations. We conclude that the B. cinerea populations in Hungary consist of a strongly recombining group II phylogenetic species.

Detection of in vivo protein interactions between Snf1-related kinase subunits with intron-tagged epitope-labelling in plants cells.

Plant orthologs of the yeast sucrose non-fermenting (Snf1) kinase and mammalian AMP-activated protein kinase (AMPK) represent an emerging class of important regulators of metabolic and stress signalling. The catalytic alpha-subunits of plant Snf1-related kinases (SnRKs) interact in the yeast two-hybrid system with different proteins that share conserved domains with the beta- and gamma-subunits of Snf1 and AMPKs. However, due to the lack of a robust technique allowing the detection of protein interactions in plant cells, it is unknown whether these proteins indeed occur in SnRK complexes in vivo. Here we describe a double-labelling technique, using intron-tagged hemagglutinin (HA) and c-Myc epitope sequences, which provides a simple tool for co-immunopurification of interacting proteins expressed in Agrobacterium-transformed Arabidopsis cells. This generally applicable plant protein interaction assay was used to demonstrate that AKINbeta2, a plant ortholog of conserved Snf1/AMPK beta-subunits, forms different complexes with the catalytic alpha-subunits of Arabidopsis SnRK protein kinases AKIN10 and AKIN11 in vivo.

Control of cell elongation and stress responses by steroid hormones and carbon catabolic repression in plants.

Molecular analysis of Arabidopsis mutants displaying hypocotyl elongation defects in both the dark and light revealed recently that steroids play an essential role as hormones in plants. Deficiencies in brassinosteroid biosynthesis and signalling permit photomorphogenic development and light-regulated gene expression in the dark, and result in severe dwarfism, male sterility and de-repression of stress-induced genes in the light. A cytochrome P450 steroid hydroxylase (CYP90) controls a rate limiting step in brassinosteroid biosynthesis and appears to function as a signalling factor in stress responses. Another key step in steroid biosynthesis is controlled by the Arabidopsis SNF1 kinases that phosphorylate the 3-hydroxy-3methylglutaryl-CoA reductase. The activity of SNF1 kinases is regulated by PRL1, an evolutionarily conserved alpha-importin-binding nuclear WD-protein. The prl1 mutation results in cell elongation defects, de-repression of numerous stress-induced genes, and augments the sensitivity of plants to glucose, cold stress and several hormones, including cytokinin, ethylene, auxin, and abscisic acid.

Pleiotropic control of glucose and hormone responses by PRL1, a nuclear WD protein, in Arabidopsis.

The prl1 mutation localized by T-DNA tagging on Arabidopsis chromosome 4-44 confers hypersensitivity to glucose and sucrose. The prl1 mutation results in transcriptional derepression of glucose responsive genes defining a novel suppressor function in glucose signaling. The prl1 mutation also augments the sensitivity of plants to growth hormones including cytokinin, ethylene, abscisic acid, and auxin; stimulates the accumulation of sugars and starch in leaves; and inhibits root elongation. PRL1 encodes a regulatory WD protein that interacts with ATHKAP2, an alpha-importin nuclear import receptor, and is imported into the nucleus in Arabidopsis. Potential functional conservation of PRL1 homologs found in other eukaryotes is indicated by nuclear localization of PRL1 in monkey COS-1 cells and selective interaction of PRL1 with a nuclear protein kinase C-betaII isoenzyme involved in human insulin signaling.

Gene identification with sequenced T-DNA tags generated by transformation of Arabidopsis cell suspension.

A protocol for establishment and high-frequency Agrobacterium-mediated transformation of morphogenic Arabidopsis cell suspensions was developed to facilitate saturation mutagenesis and identification of plant genes by sequenced T-DNA tags. Thirty-two self-circularized T-DNA tagged chromosomal loci were isolated from 21 transgenic plants by plasmid rescue and long-range inverse polymerase chain reaction (LR-iPCR). By bidirectional sequencing of the ends of T-DNA-linked plant DNA segments, nine T-DNA inserts were thus localized in genes coding for the Arabidopsis ASK1 kinase, cyclin 3b, J-domain protein, farnesyl diphosphate synthase, ORF02, an unknown EST, and homologues of a copper amine oxidase, a peripheral Golgi protein and a maize pollen-specific transcript. In addition, 16 genes were identified in the vicinity of sequenced T-DNA tags illustrating the efficiency of genome analysis by insertional mutagenesis.

A synthetic cryIC gene, encoding a Bacillus thuringiensis delta-endotoxin, confers Spodoptera resistance in alfalfa and tobacco.

Spodoptera species, representing widespread polyphagous insect pests, are resistant to Bacillus thuringiensis delta-endotoxins used thus far as insecticides in transgenic plants. Here we describe the chemical synthesis of a cryIC gene by a novel template directed ligation-PCR method. This simple and economical method to construct large synthetic genes can be used when routine resynthesis of genes is required. Chemically phosphorylated adjacent oligonucleotides of the gene to be synthesized are assembled and ligated on a single-stranded, partially homologous template derived from a wild-type gene (cryIC in our case) by a thermostable pfu DNA ligase using repeated cycles of melting, annealing, and ligation. The resulting synthetic DNA strands are selectively amplified by PCR with short specific flanking primers that are complementary only to the new synthetic DNA. Optimized expression of the synthetic cryIC gene in alfalfa and tobacco results in the production of 0.01-0.2% of total soluble proteins as CryIC toxin and provides protection against the Egyptian cotton leafworm (Spodoptera littoralis) and the beet armyworm (Spodoptera exigua). To facilitate selection and breeding of Spodoptera-resistant plants, the cryIC gene was linked to a pat gene, conferring resistance to the herbicide BASTA.

Mapping of the entomocidal fragment of Spodoptera-specific Bacillus thuringiensis toxin CryIC.

Insecticidal CryI protoxins of Bacillus thuringiensis are activated by proteolysis in the midgut of insects. A conservation of proteolytic cleavage sites in the CryI proteins facilitates the expression of active toxins in transgenic plants to obtain protection from various insects. However, the engineering of CryIC toxins has, thus far, failed to yield applicable resistance to armyworms of Spodoptera species representing common insect pests worldwide. To improve the production of recombinant CryIC toxins, we established a CryIC consensus sequence by comparative analysis of three cryIC genes and tested the stability and protease sensitivity of truncated CryIC toxins in Escherichia coli and in vitro. In contrast to previous data, the boundaries of trypsin-resistant CryIC core toxin were mapped to amino acid residues I28 and R627. Proteolysis of the truncated CryIC proteins showed that Spodoptera midgut proteases may further shorten the C-terminus of CryIC toxin to residue A615. However, C-terminal truncation of CryIC to residue L614, and a mutation causing amino acid replacement I610T, abolished the insecticidal activity of CryIC toxin to S. littoralis larvae, as well as its resistance to trypsin and Spodoptera midgut proteases. Because no CryIC toxin carrying a proteolytically processed N-terminus could be stably expressed in bacteria, our data indicate that, in contrast to other CryI proteins, an entomocidal fragment located between amino acid positions 1 and 627 is required for stable production of recombinant CryIC toxins.

Synergistic activity of a Bacillus thuringiensis delta-endotoxin and a bacterial endochitinase against Spodoptera littoralis larvae.

In an attempt to increase the insecticidal effect of the delta-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 microgram/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 micrograms of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 micrograms of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.

Brassinosteroids rescue the deficiency of CYP90, a cytochrome P450, controlling cell elongation and de-etiolation in Arabidopsis.

The cpd mutation localized by T-DNA tagging on Arabidopsis chromosome 5-14.3 inhibits cell elongation controlled by the ecdysone-like brassinosteroid hormone brassinolide. The cpd mutant displays de-etiolation and derepression of light-induced genes in the dark, as well as dwarfism, male sterility, and activation of stress-regulated genes in the light. The CPD gene encodes a cytochrome P450 (CYP90) sharing homologous domains with steroid hydroxylases. The phenotype of the cpd mutant is restored to wild type both by feeding with C23-hydroxylated brassinolide precursors and by ectopic overexpression of the CPD cDNA. Brassinosteroids also compensate for different cell elongation defects of Arabidopsis det, cop, fus, and axr2 mutants, indicating that these steroids play an essential role in the regulation of plant development.

T-DNA integration: a mode of illegitimate recombination in plants.

Transferred DNA (T-DNA) insertions of Agrobacterium gene fusion vectors and corresponding insertional target sites were isolated from transgenic and wild type Arabidopsis thaliana plants. Nucleotide sequence comparison of wild type and T-DNA-tagged genomic loci showed that T-DNA integration resulted in target site deletions of 29-73 bp. In those cases where integrated T-DNA segments turned out to be smaller than canonical ones, the break-points of target deletions and T-DNA insertions overlapped and consisted of 5-7 identical nucleotides. Formation of precise junctions at the right T-DNA border, and DNA sequence homology between the left termini of T-DNA segments and break-points of target deletions were observed in those cases where full-length canonical T-DNA inserts were very precisely replacing plant target DNA sequences. Aberrant junctions were observed in those transformants where termini of T-DNA segments showed no homology to break-points of target sequence deletions. Homology between short segments within target sites and T-DNA, as well as conversion and duplication of DNA sequences at junctions, suggests that T-DNA integration results from illegitimate recombination. The data suggest that while the left T-DNA terminus and both target termini participate in partial pairing and DNA repair, the right T-DNA terminus plays an essential role in the recognition of the target and in the formation of a primary synapsis during integration.

Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana.

A recessive pale mutation, designated as cs, was identified by transferred-DNA (T-DNA)-mediated insertional mutagenesis in Arabidopsis thaliana. The pale mutation, cosegregating with the hygromycin resistance marker of the T-DNA, was mapped to the position of the ch-42 (chlorata) locus on chromosome 4. Lack of genetic complementation between cs and ch-42 mutants indicated allelism. Plant boundaries of the T-DNA insert rescued from the pale mutant were used as probes for the isolation of genomic and full-length cDNA clones of the wild-type cs gene. Transformation of the pale mutant with T-DNA vectors carrying these clones resulted in a normal green phenotype, thus demonstrating positive complementation of the T-DNA induced mutation. DNA sequence comparison of the cs mutant and its wild-type allele revealed that the T-DNA insertion occurred 11 bp upstream of the stop codon. A fusion protein, seven amino acids longer than its wild-type counterpart of Mr 46,251, is therefore synthesized in the pale mutant. Transcript analysis during dark-light transition, in vitro protein transport assay, and the absence of DNA sequence homology between cs and known genes indicates that the light regulated expression of the cs gene results in the synthesis of a novel chloroplast protein.

High-frequency T-DNA-mediated gene tagging in plants.

An insertion element [transferred DNA (T-DNA)], transferred by soil agrobacteria into the nuclear genome of plants, was used for induction of gene fusions in Arabidopsis thaliana, Nicotiana tabacum, and Nicotiana plumbaginifolia. A promoterless aph(3')II (aminoglycoside phosphotransferase II) reporter gene was linked to the right end of the T-DNA and transformed into plants along with a plasmid replicon and a selectable hygromycin-resistance gene. Transcriptional and translational reporter gene fusions were identified by screening for APH(3')II enzyme activity in diverse tissues of transgenic plants. The frequency of gene fusions, estimated by determination of the copy number of T-DNA insertions, showed that on average 30% of T-DNA inserts induced gene fusions in Arabidopsis and Nicotiana. Gene fusions were rescued from plants by transformation of the T-DNA-linked plasmid and flanking plant DNA into Escherichia coli. By dissection of gene fusions and construction of chimeric genes, callus- and root-specific promoters were identified that showed an altered tissue specificity in the presence of a 3'-downstream-located 35S promoter. Transcript mapping of a gene fusion and expression of a non-frame transcriptional fusion of bacterial luciferase luxA and luxB genes demonstrated that dicistronic transcripts are translated in tobacco.

Activity of "nonspecific pancreatic carboxylesterase" in rat serum in experimentally induced acute pancreatitis (preliminary results).

The aim of this study was to obtain more information on the serum level of "nonspecific pancreatic carboxylesterase" (PCE) in experimentally induced acute pancreatitis in rats. The effects of caerulein stimulation, hepatic duct ligation, bile-pancreatic duct ligation or the effect of retrograde injection of saline, 5% taurocholate and sunflower oil were investigated. The activity of PCE and amylase was measured in the serum, pancreatic tissue, pancreatic juice and ascitic fluid. The changes in PCE activity were greater (both in directions to increase or decrease) than that of amylase, produced by different experimental procedures. The results confirm the thesis that the serum activity of PCE is a more sensitive diagnostic method than that of amylase to detect the inflammatory process in the pancreas or the effect of obstruction of the pancreatic duct.

Simple method for focusing x rays using tapered capillaries.

A new method of focusing x rays is described using appropriately tapered capillaries. The x rays are incident on the inner surface of the capillary below the critical glancing angle and reflect due to total external reflection. By appropriately narrowing the capillary, the x rays can thus be focused in a broad band of energies. The theory of the effect and optimum taper is described. A measurement verifying the focusing capability of the method is presented. The method appears practical for focusing bending magnet synchrotron radiation around 8 keV down to a diameter of 10 mum from an initial dimension of 1-mm(2) incident cross section with an attenuation of the total energy of ~2, i.e., an increase in the intensity per unit area of 6.5 x 10(3). Greater focusing is possible with softer x rays and from undulator sources. The wide-ranging applicability of the technique is discussed.

A simple method to transfer, integrate and study expression of foreign genes, such as chicken ovalbumin and alpha-actin in plant tumors.

A simple method for inserting foreign genes into the T-region of Agrobacterium Ti-plasmids is described. A modified cosmid (pHC 79) was introduced into a predetermined site of the T-region of pTi C58. An Agrobacterium strain harboring this modified Ti-plasmid was used as an acceptor strain into which genes, cloned in pBR322, can be introduced by mobilization from Escherichia coli. pBR322-derived plasmids cannot replicate in Agrobacterium, but can be maintained by integration into the T-region of the modified Ti-plasmids by homologous recombination. This method was used to introduce the genes for ovalbumin and alpha-actin from chicken into tobacco tumors. Southern blotting and re-isolation of the inserted genes by reverse cloning showed that the animal DNA was transferred and integrated into the plant genome without rearrangements. The alpha-actin gene is not transcribed in plant tumors, whereas transcription of the ovalbumin gene was observed, however the initiation point of transcription was different from the one used in the chicken oviduct. The RNA transcribed from the chicken ovalbumin gene is polyadenylated and ranges in size between 2 and 7 kb.