Hybridizations between formae speciales of Venturia inaequalis pave the way for a new biocontrol strategy to manage fungal plant pathogens

International audienceHybridization and adaptation to new hosts are important mechanisms of fungal disease emergence. Evaluating the risk of emergence of hybrids with enhanced virulence is then key to develop sustainable crop disease management. We evaluated this risk in Venturia inaequalis, the fungus responsible for the common and serious scab disease on Rosaceae hosts, including apple, pyracantha and loquat. Field isolates from these three hosts and progenies obtained from five crosses between formae speciales isolates collected from pyracantha (f.sp. pyracantha) and apple (f.sp. pomi) were tested for their pathogenicity on the three hosts. We confirmed a strict host specificity between isolates from apple and pyracantha, and showed that most isolates were able to cause disease on loquat. None of the 251 progeny obtained from five crosses between V. inaequalis f.sp. pyracantha and V. inaequalis f. sp. pomi could infect apple. If confirmed on more crosses, the inability of the hybrids to infect apple could lead to a novel biocontrol strategy based on a sexual hijacking of V. inaequalis f.sp. pomi by a massive introduction of V. inaequalis f.sp. pyracantha in apple orchards. This strategy, analogous to the sterile insect approach, could lead to the collapse of the population size of V. inaequalis and dramatically reduce the use of chemicals in orchards

Threat to Asian wild apple trees posed by gene flow from domesticated apple trees and their pestified pathogens

Secondary contact between crops and their wild relatives poses a threat to wild species, not only through gene flow between plants, but also through the dispersal of crop pathogens and genetic exchanges involving these pathogens, particularly those that have become more virulent by indirect selection on resistant crops, a phenomenon known as “pestification”. Joint analyses of wild and domesticated hosts and their pathogens are essential to address this issue, but such analyses remain rare. We used population genetics approaches, demographic inference and pathogenicity tests on host-pathogen pairs of wild or domesticated apple trees from Central Asia and their main fungal pathogen, Venturia inaequalis, which itself has differentiated agricultural and wild-type populations. We confirmed the occurrence of gene flow from cultivated (Malus domestica) to wild (Malus sieversii) apple trees in Asian forests, potentially threatening the persistence of Asian wild apple trees. Pathogenicity tests demonstrated the pestification of V. inaequalis, the agricultural-type population being more virulent on both wild and domesticated trees. SNP markers and the demographic modeling of pathogen populations revealed hybridization following secondary contact between agricultural and wild-type fungal populations, and dispersal of the agricultural-type pathogen population in wild forests, increasing the threat of disease in the wild apple species. We detected a SNP potentially involved in pathogen pestification, generating an early stop codon in a gene encoding a small secreted protein in the agricultural-type fungal population. Our findings, based on joint analyses of paired host and pathogen datasets, highlight the threat posed by cultivating a crop near its center of origin, in terms of pestified pathogen invasions in wild plant populations and introgression in the wild-type pathogen population

Coupling evolutionary dynamics of Venturia inaequalis effectors and functional genomic to decipher mechanisms of virulence and to identify durable resistance genes in apple.

During infection, pathogens secrete small secreted proteins (SSPs), called effectors, that promote disease. Plant receptors encoded by resistance R genes might recognize such effectors (also called avirulence factors AVRs), resulting in plant immunity. Pathogens evade recognition thanks to the emergence of virulent alleles present in populations. It has been demonstrated that avirulent effectors are crucial for the pathogen infection cycle and that their loss-of-function may induce a substantial fitness cost. This kind of effector is expected to be under purifying selective pressure. Here, we aim at identifying the effector repertoire of Venturia inaequalis, the agent of apple scab, assessing its evolutionary dynamics and studying the role of candidate effectors in virulence. We sequenced de novo 90 strains, collected on apple and on their wild relatives and differing in their host range or virulence to study allelic polymorphism at 880 putative effector loci. The top-20 hits for highly conserved sequences were selected as candidates for further functional analyses. In planta gene expression showed a significant induction of these conserved SSP at the early stage of plant infection. Their functions were investigated using targeted deletion mutants. Remarkably, loss of two conserved SSPs resulted in reduced aggressiveness without any alteration in growth in vitro. GFP-tagged protein and heterologous expression were used to assess their sub-cellular localization in infected apple leaves. Involvement of theses SSP in the modulation of host defence was also investigated using an apple full-transcript microarray. Highly conserved effectors will be used to screen for novel R genes in Malus genotypes characterized for their high resistance to scab. This combined knowledge should enable us to understand strategies used by the pathogen to overcome defences in apple and consequently to build more durable resistance towards apple scab

Coupling evolutionary dynamics of Venturia inaequalis effectors and functional genomic to decipher mechanisms of virulence and to identify durable resistance genes in apple.

During infection, pathogens secrete small secreted proteins (SSPs), called effectors, that promote disease. Plant receptors encoded by resistance R genes might recognize such effectors (also called avirulence factors AVRs), resulting in plant immunity. Pathogens evade recognition thanks to the emergence of virulent alleles present in populations. It has been demonstrated that avirulent effectors are crucial for the pathogen infection cycle and that their loss-of-function may induce a substantial fitness cost. This kind of effector is expected to be under purifying selective pressure. Here, we aim at identifying the effector repertoire of Venturia inaequalis, the agent of apple scab, assessing its evolutionary dynamics and studying the role of candidate effectors in virulence. We sequenced de novo 90 strains, collected on apple and on their wild relatives and differing in their host range or virulence to study allelic polymorphism at 880 putative effector loci. The top-20 hits for highly conserved sequences were selected as candidates for further functional analyses. In planta gene expression showed a significant induction of these conserved SSP at the early stage of plant infection. Their functions were investigated using targeted deletion mutants. Remarkably, loss of two conserved SSPs resulted in reduced aggressiveness without any alteration in growth in vitro. GFP-tagged protein and heterologous expression were used to assess their sub-cellular localization in infected apple leaves. Involvement of theses SSP in the modulation of host defence was also investigated using an apple full-transcript microarray. Highly conserved effectors will be used to screen for novel R genes in Malus genotypes characterized for their high resistance to scab. This combined knowledge should enable us to understand strategies used by the pathogen to overcome defences in apple and consequently to build more durable resistance towards apple scab

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturiapirina, Venturia aucupariae and Venturia asperata

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs

Strong differentiation within Diplocarpon rosae strains based on microsatellite markers and greenhouse‐based inoculation protocol on Rosa

The haploid ascomycete Diplocarpon rosae is the causal agent of black spot disease on roses, a widespread and devastating disease in the outdoor landscape. In this study, we established a Eurasian collection of 77 monoconidial strains of D. rosae: 50 strains collected on cultivated roses in Europe and Asia, and 27 strains on wild roses in Kazakhstan. To provide tools to describe its biology and to study its genetic diversity, we sequenced two strains of D. rosae using Illumina paired-end technology. The genome sizes of these two strains were estimated at 31.1 and 35.2 Mb, which are two times smaller than the genome size of the unique strain previously published. A BUSCO analysis confirmed a genome duplication of the strain previously sequenced and partial gene duplication of strains analysed in this study. Using the two genome sequences, 27 polymorphic microsatellite markers were identified. Polymorphism analysis of the 77 strains revealed a strong genetic differentiation between strains from cultivated and wild roses, and a lower diversity within the fungal population from cultivated roses compared to the population from wild roses. Pathogenicity of 10 strains was evaluated on 9 rose cultivars inoculated in the greenhouse. Disease scoring allowed the classification of strains into three groups and the characterization of resistance of rose cultivars. Good correlation observed between resistance scoring in greenhouse conditions and in the field indicates that pathogenicity assays in controlled conditions could be very useful in the near future to rapidly characterize the resistance of new rose varieties to black spot disease

Vaccination of squirrel monkeys (Saimiri spp.) with nanoparticle based-Toxoplasma gondii antigens: new hope for captive susceptible species

International audienceSquirrel monkeys (Saimiri spp.), new world primates from South America, are very susceptible to toxoplasmosis. Numerous outbreaks of fatal toxoplasmosis in zoos have been identified around the world, resulting in acute respiratory distress and sudden death. To date, preventive hygiene measures or available treatments are not able to significantly reduce this mortality in zoos. Therefore, vaccination seems to be the best long-term solution to control acute toxoplasmosis. Recently, we developed a nasal vaccine composed of total extract of soluble proteins of Toxoplasma gondii associated with muco-adhesive maltodextrin-nanoparticles. The vaccine, which generated specific cellular immune responses, demonstrated efficacy against toxoplasmosis in murine and ovine experimental models. In collaboration with six French zoos, our vaccine was used as a last resort in 48 squirrel monkeys to prevent toxoplasmosis. The full protocol of vaccination includes two intranasal sprays followed by combined intranasal and s.c. administration. No local or systemic side-effects were observed irrespective of the route of administration. Blood samples were collected to study systemic humoral and cellular immune responses up to 1 year after the last vaccination. Vaccination induced a strong and lasting systemic cellular immune response mediated by specific IFN-γ secretion by peripheral blood mononuclear cells. Since the introduction of vaccination, no deaths of squirrel monkeys due to T. gondii has been observed for more than 4 years suggesting the promising usage of our vaccine. Moreover, to explain the high susceptibility of naive squirrel monkeys to toxoplasmosis, their innate immune sensors were investigated. It was observed that Toll-like and Nod-like receptors appear to be functional following T. gondii recognition suggesting that the extreme susceptibility to toxoplasmosis may not be linked to innate detection of the parasite

An extended SARS-CoV-2 mRNA vaccine prime-boost interval enhances B cell immunity with limited impact on T cells

Summary: Spacing the first two doses of SARS-CoV-2 mRNA vaccines beyond 3–4 weeks raised initial concerns about vaccine efficacy. While studies have since shown that long-interval regimens induce robust antibody responses, their impact on B and T cell immunity is poorly known. Here, we compare SARS-CoV-2 naive donors B and T cell responses to two mRNA vaccine doses administered 3–4 versus 16 weeks apart. After boost, the longer interval results in a higher magnitude and a more mature phenotype of RBD-specific B cells. While the two geographically distinct cohorts present quantitative and qualitative differences in T cell responses at baseline and after priming, the second dose led to convergent features with overall similar magnitude, phenotype, and function of CD4+ and CD8+ T cell responses at post-boost memory time points. Therefore, compared to standard regimens, a 16-week interval has a favorable impact on the B cell compartment but minimally affects T cell immunity

Levels of mRNA of genes affected by thermal manipulation during embryogenesis in the <i>Pectoralis major</i> muscle of broiler chickens at d 34.

<p>Values were standardized using geNorm factor calculated from the expression of 18S ribosomal RNA, Cytochrome b and β-actin. A) DIO3: deiodinase 3; HK1: hexokinase 1; SCOT: succinyl-CoA: 3-ketoacid CoA transferase; *: <i>P</i><0.05; †: <i>P</i><0.10. B) Chickens were incubated and reared in standard conditions (Controls C), thermally manipulated during embryogenesis and reared in standard conditions (TM), or incubated in standard conditions and exposed to heat challenge at d 34 (CCh) or thermally manipulated during embryogenesis and exposed to heat challenge at d 34 (TMCh). MYOD: myoblast determination protein; GLUT 8: glucose transporter 8; PGC-1α: peroxisome-proliferator-activated receptor (PPAR) γ coactivator 1α; CS: citrate synthase; DIO2: deiodinase 2. Different letters indicate significant differences between treatments (a–b, <i>P</i><0.05) or only a tendency (A–B, <i>P</i><0.10) when both incubation and challenge (incubation) effects or challenge(incubation) effect alone were significant (n = 8 per treatment).</p