Large-scale population survey of Leptosphaeria maculans in France highlights both on-going breakdowns and potentially effective resistance genes in oilseed rape.

BACKGROUND: Leptosphaeria maculans, the cause of stem canker of oilseed rape, develops gene-for-gene interactions with its host and shows a high evolutionary potential to 'break down' novel resistance genes (R, Rlm) deployed in cultivars over large areas. For optimal management of R genes, updated knowledge of the population structure of the pathogen is needed. In France, large-scale surveys have been done at 10-year intervals since 2000. Here we report the characterization of a large L. maculans population collected in France in 2019-2020. RESULTS: A total of 844 isolates were collected from 11 sites in ten French departments and were phenotyped for their virulence against nine Brassica napus R genes. All isolates were virulent toward Rlm2 and Rlm9. Very few isolates were avirulent on Rlm1 (1.8%) and Rlm4 (0.6%). Avirulent isolates toward Rlm7 ('AvrLm7') varied from 67% to 11.3%, depending on the site sampled, illustrating the ongoing breakdown of Rlm7. The decrease of AvrLm7 isolates (29.2% at the national level) compared to the 2010 survey (96.5%) was accompanied by an increase of avirulent isolates on Rlm3 (0% in 2010; 54% in 2019-2020). However, virulent isolates on both Rlm3 and Rlm7, previously rarely detected, were found in all sites with a frequency of 17.3%. Finally, most or all isolates were avirulent on Rlm11 (96.1%), LepR2 (RlmS, 99.8%), and Rlm6 (100%), suggesting these three genes still effectively control the disease. CONCLUSION: These data will help guide strategies for breeding and deploying resistant oilseed rape varieties against L. maculans in France. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Location and timing govern tripartite interactions of fungal phytopathogens and host in the stem canker species complex.

BACKGROUND: Leptosphaeria maculans "brassicae" (Lmb) and Leptosphaeria biglobosa "brassicae" (Lbb) make up a species complex involved in the stem canker (blackleg) disease of rapeseed (Brassica napus). They coinfect rapeseed together, from the early stage of infection on leaves to the final necrotic stage at the stem base, and both perform sexual crossings on plant residues. L. biglobosa is suggested to be a potential biocontrol agent against Lmb, but there has been no mechanistic investigation of the different types of interactions that may occur between the plant and the two fungal species. RESULTS: We investigated the bi- or tripartite interaction mechanisms by (i) confronting Lmb and Lbb in culture conditions or during cotyledon infection, with different timing and/or spore concentration regimes, (ii) performing RNA-Seq experiments in vitro or on the kinetics of infection of cotyledons infected by Lmb and/or Lbb to evaluate the transcriptomic activity and the plant response when both fungal species are inoculated together. Lbb infection of B. napus cotyledons was typical of a necrotrophic behavior, with a very early setup of one pathogenicity program and very limited colonization of tissues. This contrasted with the complex succession of pathogenicity programs of the hemibiotroph Lmb. During simultaneous co-infection by both species, Lmb was strongly impacted in its growth and transcriptomic dynamics both in vitro and in planta, while Lbb was unaffected by the presence of Lmb. However, the drastic inhibition of Lmb growth by Lbb was ineffective in the case of delayed inoculation with Lbb or a lower amount of spores of Lbb compared to Lmb. CONCLUSIONS: Our data suggest that Lmb growth inhibition by Lbb is the result of a combination of factors that may include competition for trophic resources, the generation by Lbb of an environment unsuitable for the lifecycle of Lmb or/and the effect on Lmb of plant defense responses induced by Lbb. It indicates that growth inhibition occurs in very specific conditions (i.e., co-inoculation at the same place of an equal amount of inoculum) that are unlikely to occur in the field where their coexistence does not prevent any species from completing their life cycle.

Polymorphism of Avirulence Genes and Adaptation to Brassica Resistance Genes Is Gene-Dependent in the Phytopathogenic Fungus Leptosphaeria maculans.

The fungal phytopathogen Leptosphaeria maculans, which causes stem canker (blackleg) of rapeseed (Brassica napus), is mainly controlled worldwide by genetic resistance, which includes major resistance genes (Rlm). This model is one of those for which the highest number of avirulence genes (AvrLm) has been cloned. In many systems, including the L. maculans-B. napus interaction, intense use of resistance genes exerts strong selection pressure on the corresponding avirulent isolates, and the fungi may rapidly escape resistance through various molecular events which modify the avirulence genes. In the literature, the study of polymorphism at avirulence loci is often focused on single genes under selection pressure. In this study, we investigate allelic polymorphism at 11 avirulence loci in a French population of 89 L. maculans isolates collected on a trap cultivar in four geographic locations in the 2017-2018 cropping season. The corresponding Rlm genes have been (i) used for a long time, (ii) recently used, or (iii) unused in agricultural practice. The sequence data generated indicate an extreme diversity of situations. For example, genes submitted to an ancient selection may have either been deleted in populations (AvrLm1) or replaced by a single-nucleotide mutated virulent version (AvrLm2, AvrLm5-9). Genes that have never been under selection may either be nearly invariant (AvrLm6, AvrLm10A, AvrLm10B), exhibit rare deletions (AvrLm11, AvrLm14), or display a high diversity of alleles and isoforms (AvrLmS-Lep2). These data suggest that the evolutionary trajectory of avirulence/virulence alleles is gene-dependent and independent of selection pressure in L. maculans. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Impact of a resistance gene against a fungal pathogen on the plant host residue microbiome: The case of the Leptosphaeria maculans-Brassica napus pathosystem.

Oilseed rape residues are a crucial determinant of stem canker epidemiology as they support the sexual reproduction of the fungal pathogen Leptosphaeria maculans. The aim of this study was to characterize the impact of a resistance gene against L. maculans infection on residue microbial communities and to identify microorganisms interacting with this pathogen during residue degradation. We used near-isogenic lines to obtain healthy and infected host plants. The microbiome associated with the two types of plant residues was characterized by metabarcoding. A combination of linear discriminant analysis and ecological network analysis was used to compare the microbial communities and to identify microorganisms interacting with L. maculans. Fungal community structure differed between the two lines at harvest, but not subsequently, suggesting that the presence/absence of the resistance gene influences the microbiome at the base of the stem whilst the plant is alive, but that this does not necessarily lead to differential colonization of the residues by fungi. Direct interactions with other members of the community involved many fungal and bacterial amplicon sequence variants (ASVs). L. maculans appeared to play a minor role in networks, whereas one ASV affiliated to Plenodomus biglobosus (synonym Leptosphaeria biglobosa) from the Leptosphaeria species complex may be considered a keystone taxon in the networks at harvest. This approach could be used to identify and promote microorganisms with beneficial effects against residue-borne pathogens and, more broadly, to decipher the complex interactions between multispecies pathosystems and other microbial components in crop residues.

Major changes in grapevine wood microbiota are associated with the onset of esca, a devastating trunk disease.

Esca, a major grapevine trunk disease in old grapevines, is associated with the colonization of woody tissues by a broad range of plant pathogenic fungi. To identify which fungal and bacterial species are involved in the onset of this disease, we analysed the microbiota from woody tissues of young (10-year-old) grapevines at an early stage of esca. Using meta-barcoding, 515 fungal and 403 bacterial operational taxonomic units (OTUs) were identified in woody tissues. In situ hybridization showed that these fungi and bacteria co-inhabited in grapevine woody tissues. In non-necrotic woody tissues, fungal and bacterial microbiota varied according to organs and seasons but not diseased plant status. Phaeomoniella chlamydospora, involved in the Grapevine trunk disease, was the most abundant species in non-necrotic tissues from healthy plants, suggesting a possible non-pathogenic endophytic behaviour. Most diseased plants (70%) displayed cordons, with their central white-rot necrosis colonized essentially by two plant pathogenic fungi (Fomitiporia mediterranea: 60%-90% and P. chlamydospora: 5%-15%) and by a few bacterial taxa (Sphingomonas spp. and Mycobacterium spp.). The occurrence of a specific association of fungal and bacterial species in cordons from young grapevines expressing esca-foliar symptoms strongly suggests that that microbiota is involved in the onset of this complex disease.

Differential dynamics of microbial community networks help identify microorganisms interacting with residue-borne pathogens: the case of Zymoseptoria tritici in wheat.

BACKGROUND: Wheat residues are a crucial determinant of the epidemiology of Septoria tritici blotch, as they support the sexual reproduction of the causal agent Zymoseptoria tritici. We aimed to characterize the effect of infection with this fungal pathogen on the microbial communities present on wheat residues and to identify microorganisms interacting with it. We used metabarcoding to characterize the microbiome associated with wheat residues placed outdoors, with and without preliminary Z. tritici inoculation, comparing the first set of residues in contact with the soil and a second set without contact with the soil, on four sampling dates in two consecutive years. RESULTS: The diversity of the tested conditions, leading to the establishment of different microbial communities according to the origins of the constitutive taxa (plant only, or plant and soil), highlighted the effect of Z. tritici on the wheat residue microbiome. Several microorganisms were affected by Z. tritici infection, even after the disappearance of the pathogen. Linear discriminant analyses and ecological network analyses were combined to describe the communities affected by the infection. The number of fungi and bacteria promoted or inhibited by inoculation with Z. tritici decreased over time and was smaller for residues in contact with the soil. The interactions between the pathogen and other microorganisms appeared to be mostly indirect, despite the strong position of the pathogen as a keystone taxon in networks. Direct interactions with other members of the communities mostly involved fungi, including other wheat pathogens. Our results provide essential information about the alterations to the microbial community in wheat residues induced by the mere presence of a fungal pathogen, and vice versa. Species already described as beneficial or biocontrol agents were found to be affected by pathogen inoculation. CONCLUSIONS: The strategy developed here can be viewed as a proof-of-concept focusing on crop residues as a particularly rich ecological compartment, with a high diversity of fungal and bacterial taxa originating from both the plant and soil compartments, and for Z. tritici-wheat as a model pathosystem. By revealing putative antagonistic interactions, this study paves the way for improving the biological control of residue-borne diseases.

Crop Residues in Wheat-Oilseed Rape Rotation System: a Pivotal, Shifting Platform for Microbial Meetings.

Crop residues are a crucial ecological niche with a major biological impact on agricultural ecosystems. In this study, we used a combined diachronic and synchronic field experiment based on wheat-oilseed rape rotations to test the hypothesis that plant is a structuring factor of microbial communities in crop residues, and that this effect decreases over time with their likely progressive degradation and colonisation by other microorganisms. We characterised an entire fungal and bacterial community associated with 150 wheat and oilseed rape residue samples at a plurennial scale by metabarcoding. The impact of plant species on the residue microbiota decreased over time and our data revealed turnover, with the replacement of oligotrophs, often plant-specific genera (such as pathogens) by copiotrophs, belonging to more generalist genera. Within a single cropping season, the plant-specific genera and species were gradually replaced by taxa that are likely to originate from the soil. These changes occurred more rapidly for bacteria than for fungi, known to degrade complex compounds. Overall, our findings suggest that crop residues constitute a key fully-fledged microbial ecosystem. Taking into account this ecosystem, that has been neglected for too long, is essential, not only to improve the quantitative management of residues, the presence of which can be detrimental to crop health, but also to identify groups of beneficial microorganisms. Our findings are of particular importance, because the wheat-oilseed rape rotation, in which no-till practices are frequent, is particularly widespread in the European arable cropping systems.

Metabarcoding targeting the EF1 alpha region to assess Fusarium diversity on cereals.

Fusarium head blight (FHB) is a major cereal disease caused by a complex of Fusarium species. These species vary in importance depending on climatic conditions, agronomic factors or host genotype. In addition, Fusarium species can release toxic secondary metabolites. These mycotoxins constitute a significant food safety concern as they have health implications in both humans and animals. The Fusarium species involved in FHB differ in their pathogenicity, ability to produce mycotoxins, and fungicide sensitivity. Accurate and exhaustive identification of Fusarium species in planta is therefore of great importance. In this study, using a new set of primers targeting the EF1α gene, the diversity of Fusarium species on cereals was evaluated using Illumina high-throughput sequencing. The PCR amplification parameters and bioinformatic pipeline were optimized with mock and artificially infected grain communities and further tested on 65 field samples. Fusarium species were retrieved from mock communities and good reproducibility between different runs or PCR cycle numbers was be observed. The method enabled the detection of as few as one single Fusarium-infected grain in 10,000. Up to 17 different Fusarium species were detected in field samples of barley, durum and soft wheat harvested in France. This new set of primers enables the assessment of Fusarium diversity by high-throughput sequencing on cereal samples. It provides a more exhaustive picture of the Fusarium community than the currently used techniques based on isolation or species-specific PCR detection. This new experimental approach may be used to show changes in the composition of the Fusarium complex or to detect the emergence of new Fusarium species as far as the EF1α sequence of these species show a sufficient amount of polymorphism in the portion of sequence analyzed. Information on the distribution and prevalence of the different Fusarium species in a given geographical area, and in response to various environmental factors, is of great interest for managing the disease and predicting mycotoxin contamination risks.

Mutual Exclusion between Fungal Species of the Fusarium Head Blight Complex in a Wheat Spike.

Fusarium head blight (FHB) is one of the most damaging diseases of wheat. FHB is caused by a species complex that includes two genera of Ascomycetes: Microdochium and Fusarium. Fusarium graminearum, Fusarium culmorum, Fusarium poae, and Microdochium nivale are among the most common FHB species in Europe and were chosen for these experiments. Field studies and surveys show that two or more species often coexist within the same field or grain sample. In this study, we investigated the competitiveness of isolates of different species against isolates of F. graminearum at the scale of a single spike. By performing point inoculations of a single floret, we ensured that each species was able to establish independent infections and competed for spike colonization only. The fungal colonization was assessed in each spike by quantitative PCR. After establishing that the spike colonization was mainly downwards, we compared the relative colonization of each species in coinoculations. Classical analysis of variance suggested a competitive interaction but remained partly inconclusive because of a large between-spike variance. Further data exploration revealed a clear exclusion of one of the competing species and the complete absence of coexistence at the spike level.

Interactions between head blight pathogens: consequences for disease development and toxin production in wheat spikes.

Head blight (HB) is one of the most damaging diseases on wheat, inducing significant yield losses and toxin accumulation in grains. Fungal pathogens responsible for HB include the genus Microdochium, with two species, and the toxin producer genus Fusarium, with several species. Field studies and surveys show that two or more species can coexist within a same field and coinfect the same plant or the same spike. In the current study, we investigated how the concomitant presence of F. graminearum and another of the HB complex species influences the spike colonization and the toxin production by the fungi. To study these interactions, 17 well-characterized isolates representing five species were inoculated alone or in pairs on wheat spikes in greenhouse and field experiments. The fungal DNA in the grains was estimated by quantitative PCR and toxin contents (deoxynivalenol and nivalenol) by ultraperformance liquid chromatography-UV detection-tandem mass spectrometry. The responses of the different isolates to the presence of a competitor were variable and isolate specific more than species specific. The development of the most aggressive isolates was either unchanged or a slightly increased, while the development of the less aggressive isolates was reduced. The main outcome of the study was that no trend of increased toxin production was observed in coinoculations compared to single inoculations. On the contrary, the amount of toxin produced was often lower than expected in coinoculations. We thus conclude against the hypothesis that the co-occurrence of several HB-causing species in the same field might aggravate the risk linked to fusarium toxins in wheat production.

A high-throughput multiplex method adapted for GMO detection.

A high-throughput multiplex assay for the detection of genetically modified organisms (GMO) was developed on the basis of the existing SNPlex method designed for SNP genotyping. This SNPlex assay allows the simultaneous detection of up to 48 short DNA sequences (approximately 70 bp; "signature sequences") from taxa endogenous reference genes, from GMO constructions, screening targets, construct-specific, and event-specific targets, and finally from donor organisms. This assay avoids certain shortcomings of multiplex PCR-based methods already in widespread use for GMO detection. The assay demonstrated high specificity and sensitivity. The results suggest that this assay is reliable, flexible, and cost- and time-effective for high-throughput GMO detection.

Development of a real-time PCR method for the differential detection and quantification of four solanaceae in GMO analysis: potato (Solanum tuberosum), tomato (Solanum lycopersicum), eggplant (Solanum melongena), and pepper (Capsicum annuum).

The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.

A strategy for designing multi-taxa specific reference gene systems. example of application--ppi phosphofructokinase (ppi-PPF) used for the detection and quantification of three taxa: maize (Zea mays), cotton (Gossypium hirsutum) and rice (Oryza sativa).

In the first part of the paper, we report the description of a new strategy for the development of a plant reference gene system that can be used for genetically modified organism (GMO) analysis. On the basis of in silico research for candidate genes, the design of degenerate primers allowed the obtention of genomic sequences of the selected gene ppi-phosphofructokinase ( ppi-PPF) for nine taxa in which GMOs have been developed. The comparison and the analysis of inter- and intraspecies sequence variability were performed using a large number of species and cultivars. As an example of application following the detection of single nucleotide polymorphism, we designed specific conventional and real-time polymerase chain reaction tests for the detection and quantification of three taxa, namely, maize, cotton, and rice. This system was highly specific and sensitive. The gene copy number conservation among different cultivars was analyzed and confirmed with a sequencing step. This reference gene system is adequate for use in routine assays for the quantification of GMOs. We then explain briefly the constraints faced and propose recommendations when designing a reference gene system depending on the species to be targeted.

Long-distance movement of Cauliflower mosaic virus and host defence responses in Arabidopsis follow a predictable pattern that is determined by the leaf orthostichy.

Long-distance virus transport takes place through the vascular system and is dependent on the movement of photoassimilates. Here, patterns of symptom development, virus movement and gene expression were analysed in Arabidopsis following inoculation with Cauliflower mosaic virus (CaMV) on a single leaf. Virus accumulation and expression of markers for the salicylic acid (SA) and ethylene/jasmonate (Et/JA) defence pathways, PR-1 and PDF1.2, were analysed on a leaf-by-leaf basis by real-time reverse transcription polymerase chain reaction (qRT-PCR). Virus spread followed a strictly defined pattern identical to that of a source-sink relationship. This was exploited to study differences between local and systemic defence responses in a developmental and spatial manner. In infected plants, PR-1 transcripts accumulated primarily but not exclusively in leaves with a direct vascular connection to the inoculated leaf. Abundances fell significantly as virus accumulated. By contrast, PDF1.2 transcripts were significantly lower than in controls in all leaves at early stages of infection, but recovered as virus accumulated. Virus and PR-1 transcript abundances are negatively correlated, and SA- and Et/JA-mediated signalling of gene expression occurs independently of the presence of virus. Although SA-dependent signalling responses were mainly linked to the orthostichy, Et/JA-dependent responses were independent of vascular connections.

Components of Arabidopsis defense- and ethylene-signaling pathways regulate susceptibility to Cauliflower mosaic virus by restricting long-distance movement.

We analyzed the susceptibility of Arabidopsis mutants with defects in salicylic acid (SA) and jasmonic acid (JA)/ethylene (ET) signaling to infection by Cauliflower mosaic virus (CaMV). Mutants cpr1-1 and cpr5-2, in which SA-dependent defense signaling is activated constitutively, were substantially more resistant than the wild type to systemic infection, implicating SA signaling in defense against CaMV. However, SA-deficient NahG, sid2-2, eds5-1, and pad4-1 did not show enhanced susceptibility. A cpr5 eds5 double mutant also was resistant, suggesting that resistance in cpr5 may function partially independently of SA. Treatment of cpr5 and cpr5 eds5, but not cpr1, with salicyl-hydroxamic acid, an inhibitor of alternative oxidase, partially restored susceptibility to wild-type levels. Mutants etr1-1, etr1-3, and ein2-1, and two mutants with lesions in ET/JA-mediated defense, eds4 and eds8, also showed reduced virus susceptibility, demonstrating that ET-dependent responses also play a role in susceptibility. We used a green fluorescent protein (GFP)-expressing CaMV recombinant to monitor virus movement. In mutants with reduced susceptibility, cpr1-1, cpr5-2, and etr1-1, CaMV-GFP formed local lesions similar to the wild type, but systemic spread was almost completely absent in cpr1 and cpr5 and was substantially reduced in etr1-1. Thus, mutations with enhanced systemic acquired resistance or compromised ET signaling show diminished long-distance virus movement.

Cauliflower mosaic virus, a compatible pathogen of Arabidopsis, engages three distinct defense-signaling pathways and activates rapid systemic generation of reactive oxygen species.

We analyzed expression of marker genes for three defense pathways during infection by Cauliflower mosaic virus (CaMV), a compatible pathogen of Arabidopsis (Arabidopsis thaliana), using luciferase reporter transgenes and directly by measuring transcript abundance. Expression of PR-1, a marker for salicylic acid signaling, was very low until 8 d postinoculation and then rose sharply, coinciding with the rise in virus levels. In contrast, as early as 2 h postinoculation, transcriptional up-regulation of GST1-a marker for reactive oxygen species-and PDF1.2-a marker for jasmonic acid/ethylene defense signaling-was detectable in the virus-inoculated leaf and systemically. In parallel with the activation of GST1, H(2)O(2) accumulated locally and systemically in virus- but not mock-inoculated plants. However, in plants inoculated with infectious CaMV DNA rather than virus particles, the onset of systemic luciferase activity was delayed by 24 to 48 h, suggesting that virion structural proteins act as the elicitor. This phenomenon, which we term the rapid systemic response, preceded virus movement from the inoculated leaf; therefore, the systemic signal is not viral. Systemic, but not local, H(2)O(2) accumulation was abolished in rbohDF double mutants and in etr1-1 and ein2-1 mutants, implicating NADPH oxidase and ethylene signaling in the generation and transduction of the response. Ethylene, but not rbohDF mutants, also showed reduced susceptibility to CaMV, whereas in NahG transgenics, virus levels were similar to wild type. These findings implicate reactive oxygen species and ethylene in signaling in response to CaMV infection, but suggest that salicylic acid does not play an effective role.

Inappropriate annotation of a key defence marker in Arabidopsis: will the real PR-1 please stand up?

PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two "full genome" Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as "PR-1-like". Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as " PR-1" is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.

Seed germination is blocked in Arabidopsis putative vacuolar sorting receptor (atbp80) antisense transformants.

The membrane receptor protein from pea, peabp80, has been shown to function by in vitro binding studies, and in vivo in yeast mutant, as a vacuolar sorting receptor (VSR). Families of proteins with homology to peabp80 have been identified in many plants including Arabidopsis: The family of membrane receptors, atbp80a-f (Arabidopsis thaliana binding protein 80 kDa) is highly homologous to peabp80 and may also function as vacuolar sorting receptors. Interactions with vacuolar sorting determinants have been shown only in vitro for atbp80b. In this paper, atbp80b was over- and under-expressed in Arabidopsis. Transgenic plants that over-expressed atbp80b showed no visible phenotype. However, antisense transformants were defective in germination. In non-germinating antisense transformants the embryo appeared to be normal, but, using several methods, it was not possible to rescue the non-germinating seeds, indicating that the mechanisms were probably independent of a seed-coat-imposed inhibition. To make a correlation between the lack of germination and gene expression, transcription analysis of all atbp80 genes was performed in the non-germinating antisense seeds indicating that all the normally transcribed genes were not detected. Then, a gene expression study of atbp80s genes was carried-out following seed imbibition and in various organs during wild-type plant development showing that all the genes from the family were transcribed and differentially expressed.

Distribution of actin gene isoforms in the Arabidopsis leaf measured in microsamples from intact individual cells.

The contents of single plant cells can be sampled using glass microcapillaries. By combining such single-cell sampling with reverse transcription-polymerase chain reaction (RT-PCR), transcripts of individual genes can be identified and, in principle, quantified. This provides a valuable technique for the analysis and quantification of the intercellular distribution of gene expression in complex tissues. In a proof-of-principle study, the cellular locations of the transcripts of the eight isoforms of actin ( ACT) expressed in Arabidopsis thaliana (L.) Heynh. were analyzed. Cell sap was extracted from epidermal and mesophyll cells of leaves of 3- to 4-week-old plants. Single-cell (SC)-RT-PCR was used to amplify the actin transcripts using specific primer pairs for ACT1, 2, 3, 4, 7, 8, 11 and 12. Only ACT2 and ACT8 were found in epidermal and in mesophyll cells. In individual trichomes, in addition to ACT2 and ACT8, ACT7 and ACT11 transcripts were detectable. By employing the already well-characterized actin system we demonstrate the practicality and power of SC-RT-PCR as a technique for analyzing gene expression at the ultimate level of resolution, the single cell.