Human IRF1 governs macrophagic IFN-γ immunity to mycobacteria

Inborn errors of human IFN-γ-dependent macrophagic immunity underlie mycobacterial diseases, whereas inborn errors of IFN-α/β-dependent intrinsic immunity underlie viral diseases. Both types of IFNs induce the transcription factor IRF1. We describe unrelated children with inherited complete IRF1 deficiency and early-onset, multiple, life-threatening diseases caused by weakly virulent mycobacteria and related intramacrophagic pathogens. These children have no history of severe viral disease, despite exposure to many viruses, including SARS-CoV-2, which is life-threatening in individuals with impaired IFN-α/β immunity. In leukocytes or fibroblasts stimulated in vitro, IRF1-dependent responses to IFN-γ are, both quantitatively and qualitatively, much stronger than those to IFN-α/β. Moreover, IRF1-deficient mononuclear phagocytes do not control mycobacteria and related pathogens normally when stimulated with IFN-γ. By contrast, IFN-α/β-dependent intrinsic immunity to nine viruses, including SARS-CoV-2, is almost normal in IRF1-deficient fibroblasts. Human IRF1 is essential for IFN-γ-dependent macrophagic immunity to mycobacteria, but largely redundant for IFN-α/β-dependent antiviral immunity

Etudes des mécanismes moléculaires impliqués dans la transmission des agents pathogènes aux semences d'Arabidopsis thaliana

Seed transmission is a key stage of infectious cycle in diseases epidemiology of seed-borne pathogens and allows seed-borne pathogens to survive and ensure their dispersion. In order to study mechanisms involved in seed transmission two model pathosystems have been developed to get rid of the randomness inherent in field experiments. Two pathogens, a bacteria (Xanthomonas campestris pv. campestris, Xcc) and a fungus (Alternaria brassicicola) were selected to study their transmission to seeds of Arabidopsis thaliana. Microscopic observations allowed us to better analyze histological interactions during seed transmission process. Vascular tissues colonization of scape by Xcc was visualized by confocal laser scanning microscopy and revealed that Xcc is confined in xylem tissues. Electron microscopy identified the different pathways of A. brassicicola silique penetration and seed colonization. Concerning Xcc, one (Xcc2828, xytA) of two tested Ton-B dependant transporters is involved in siliques and seeds colonization but is not involved in xylem colonization, indicating a propable specificity of this transporter with primary wall xylans. We also showed that two sigma54 factors differentially regulated the expression of genes linked to mobility and biofilm formation in Xcc, which constitute essential processes for seed transmission. Concerning A. brassicicola, seed colonization requires a functional osmosensor, the group III histidine kinase AbNIK1. Finally, proteins, potentially acting in the cell as molecular chaperones, seems to be involved in the transmission of pathogens to A. thaliana seeds both in Xcc (with harpins proteins) and in A. brassicicola (with dehydrins).La transmission à et par la semence correspond à une étape critique dans l'épidémiologie de nombreuses maladies et permet aux microorganismes pathogènes de survivre et d'assurer leur dispersion. Afin d'étudier les mécanismes impliqués dans la transmission à et par la semence, deux pathosystèmes modèles ont été mis au point. Deux agents pathogènes, une bactérie (Xanthomonas campestris pv. campestris), et un champignon (Alternaria brassicicola) ont été sélectionnés pour étudier leur transmission à et par la graine de la plante hôte modèle Arabidopsis thaliana. Nous avons montré par microscopie confocale à balayage laser que la colonisation de la hampe florale par X. campestris pv. campestris est strictement confinée aux tissus vasculaires du xylème. Une approche par microscopie électronique à balayage a permis d'identifier les différentes voies de pénétration d'A. brassicicola dans la silique d'A. thaliana et le mode de colonisation des graines. L'un (Xcc2828, xytA) des deux transporteurs TonB-dépendants testés intervient dans la colonisation des valves et graines mais pas dans la colonisation du xylème de la hampe florale montrant une probable spécificité de ce TBDT pour les xylanes des parois primaires. Nous avons montré que deux facteurs o54 (Xcc1935 Xcc2802) régulent différentiellement l'expression de gènes impliqués dans la mobilité et la formation de bio ilms chez X. campestris pv. Campestris, fonctions indispensables à la transmission à et par la graine. D'autre part, une histidine kinase de groupe III osmosenseur, la proteine AbNik1, intervient lors de la colonisation des graines d'A. thaliana par A. brassicicola

Study of molecular mechanisms involved in pathogens transmission to Arabidopsis thaliana seeds.

Seed transmission is a key stage of infectious cycle in diseases epidemiology of seed-borne pathogens and allows seed-borne pathogens to survive and ensure their dispersion. In order to study mechanisms involved in seed transmission two model pathosystems have been developed to get rid of the randomness inherent in field experiments. Two pathogens, a bacteria (Xanthomonas campestris pv. campestris, Xcc) and a fungus (Alternaria brassicicola) were selected to study their transmission to seeds of Arabidopsis thaliana. Microscopic observations allowed us to better analyze histological interactions during seed transmission process. Vascular tissues colonization of scape by Xcc was visualized by confocal laser scanning microscopy and revealed that Xcc is confined in xylem tissues. Electron microscopy identified the different pathways of A. brassicicola silique penetration and seed colonization. Concerning Xcc, one (Xcc2828, xytA) of two tested Ton-B dependant transporters is involved in siliques and seeds colonization but is not involved in xylem colonization, indicating a propable specificity of this transporter with primary wall xylans. We also showed that two sigma54 factors differentially regulated the expression of genes linked to mobility and biofilm formation in Xcc, which constitute essential processes for seed transmission. Concerning A. brassicicola, seed colonization requires a functional osmosensor, the group III histidine kinase AbNIK1. Finally, proteins, potentially acting in the cell as molecular chaperones, seems to be involved in the transmission of pathogens to A. thaliana seeds both in Xcc (with harpins proteins) and in A. brassicicola (with dehydrins).La transmission à et par la semence correspond à une étape critique dans l'épidémiologie de nombreuses maladies et permet aux microorganismes pathogènes de survivre et d'assurer leur dispersion. Afin d'étudier les mécanismes impliqués dans la transmission à et par la semence, deux pathosystèmes modèles ont été mis au point. Deux agents pathogènes, une bactérie (Xanthomonas campestris pv. campestris), et un champignon (Alternaria brassicicola) ont été sélectionnés pour étudier leur transmission à et par la graine de la plante hôte modèle Arabidopsis thaliana. Nous avons montré par microscopie confocale à balayage laser que la colonisation de la hampe florale par X. campestris pv. campestris est strictement confinée aux tissus vasculaires du xylème. Une approche par microscopie électronique à balayage a permis d'identifier les différentes voies de pénétration d'A. brassicicola dans la silique d'A. thaliana et le mode de colonisation des graines. L'un (Xcc2828, xytA) des deux transporteurs TonB-dépendants testés intervient dans la colonisation des valves et graines mais pas dans la colonisation du xylème de la hampe florale montrant une probable spécificité de ce TBDT pour les xylanes des parois primaires. Nous avons montré que deux facteurs o54 (Xcc1935 Xcc2802) régulent différentiellement l'expression de gènes impliqués dans la mobilité et la formation de bio ilms chez X. campestris pv. Campestris, fonctions indispensables à la transmission à et par la graine. D'autre part, une histidine kinase de groupe III osmosenseur, la proteine AbNik1, intervient lors de la colonisation des graines d'A. thaliana par A. brassicicola

Dehydrin-like proteins in the necrotrophic fungus Alternaria brassicicola have a role in plant pathogenesis and stress response.

In this study, the roles of fungal dehydrin-like proteins in pathogenicity and protection against environmental stresses were investigated in the necrotrophic seed-borne fungus Alternaria brassicicola. Three proteins (called AbDhn1, AbDhn2 and AbDhn3), harbouring the asparagine-proline-arginine (DPR) signature pattern and sharing the characteristic features of fungal dehydrin-like proteins, were identified in the A. brassicicola genome. The expression of these genes was induced in response to various stresses and found to be regulated by the AbHog1 mitogen-activated protein kinase (MAPK) pathway. A knock-out approach showed that dehydrin-like proteins have an impact mainly on oxidative stress tolerance and on conidial survival upon exposure to high and freezing temperatures. The subcellular localization revealed that AbDhn1 and AbDhn2 were associated with peroxisomes, which is consistent with a possible perturbation of protective mechanisms to counteract oxidative stress and maintain the redox balance in AbDhn mutants. Finally, we show that the double deletion mutant ΔΔabdhn1-abdhn2 was highly compromised in its pathogenicity. By comparison to the wild-type, this mutant exhibited lower aggressiveness on B. oleracea leaves and a reduced capacity to be transmitted to Arabidopsis seeds via siliques. The double mutant was also affected with respect to conidiation, another crucial step in the epidemiology of the disease

Alignment of the repeated DPR domains of AbDhn1, AbDhn2 and AbDhn3.

<p>Conserved amino acids are boxed in black (identical) or grey (similar). DHN1.1–DHN1.2 designate the two domains from AbDhn1, DHN2.1–DHN2.5 the five domains of AbDhn2, and DHN3.1–DHN3.3 the three domains from AbDhn3. Numbers indicate the amino acid positions. The conserved DPR motif is boxed in red.</p

Transmission capacity of <i>A. brassicicola</i> wild-type (WT) and AbDhn-deficient genotypes to <i>Arabidopsis thaliana</i> seeds (L<i>er</i> ecotype).

<p>The seed transmission capacity according to the silique stage and global seed transmission capacity (strain model) were measured as described by Pochon et al <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075143#pone.0075143-Pochon1" target="_blank">[26]</a>. The five youngest siliques of at least five plants were inoculated with each fungal genotype and the experiment was repeated twice. Contaminated siliques were harvested 10 dpi. After dissection, seeds were incubated separately on PDA medium for 2 days. A seed was considered contaminated when incubation resulted in typical <i>A. brassicicola</i> colony development. For each inoculated fungal genotype, the seed infection probability was evaluated from at least 1000 seeds. Values represent infection probabilities with 95% confidence interval.</p

Susceptibility of AbDhn-deficient mutants to temperature stress.

<p>Calibrated water suspensions of conidia from the wild-type (WT) strain <i>Abra43</i> and AbDhn-deficient mutants were left for 10 h at various temperatures (−20°C, +4°C, +20°C, +40°C). Conidia were then used to inoculate microplate wells and nephelometric growth curves were established over a 33 h period. ΔLag time was calculated as the difference between the lag time at the tested temperature and the lag time at 20°C and was used as a parameter to estimate the effect of the treatment on spore viability. Error bars indicate standard deviations and asterisks indicate values that are significantly (<i>P</i><0.01) higher than that of the wild-type. Each genotype was analysed in triplicate and the experiments were repeated twice times per growth condition.</p

Subcellular localization of the AbDhn1-GFP fusion protein.

<p>Double-labelled strains expressing AbDhn1<i>-</i>GFP and DsRed-SKL were exposed to 350 mM NaCl for 2 h. Co-localization analyses in conidia (A) and hyphae (B) were examined using confocal microscopy. Bars = 10 µm.</p

Characteristics of the dehydrin-like proteins found in <i>A. brassicicola.</i>

*<p>values indicate percentage of total amino acids.</p

Expression of AbDhn-GFP proteins under salt stress.

<p>Germinated conidia (24 h-old) from strains expressing AbDhn-GFP fusions under the control of their own promoters or from a strain constitutively expressing GFP (cGFP) were either exposed to 350 mM NaCl for 2 h or left without stress. Proteins extracts were immunoblotted with HRP-coupled antibodies directed against GFP. Numbers correspond to molecular weights in kDa. The two AbDhn2 isoforms are indicated.</p