La familia génica FTF determina el patrón de colonización y la virulencia en Fusarium oxysporum

[ES] La familia génica FTF (Fusarium Transcription Factor) comprende un gen de copia única, FTF2, que está presente en todos los ascomicetos filamentosos analizados, y varias copias de FTF1, que es exclusivo de Fusarium oxysporum. Un sistema de silenciamiento de genes mediado por ARN ha sido desarrollado para atenuar específicamente los ARNm producidos por todos los genes FTF, probándose en dos formae speciales: F. oxysporum f. sp. phaseoli (cuyo hospedador es la judía) y F. oxysporum f. sp. lycopersici (cuyo hospedador es el tomate). La disminución de la expresión de FTF da lugar a una marcada reducción en la virulencia, una expresión reducida de varios genes SIX (Secreted In Xylem), la familia de efectores mejor estudiada en F. oxysporum, y menores niveles de ARNm de SGE1 (SIX Gene Expression 1), el presunto regulador de la expresión de algunos genes SIX. Por otra parte, los mutantes silenciados muestran un patrón de colonización de la planta huésped similar al mostrado por las estirpes carentes de copias de FTF1 (estirpes poco virulentas), caracterizado por una colonización de los haces xilemáticos lenta y de baja eficiencia, y una fuerte y temprana inducción de la resistencia sistémica adquirida (SAR) en el huésped

Utilizing volatile organic compounds for early detection of Fusarium circinatum

Fusarium circinatum, a fungal pathogen deadly to many Pinus species, can cause significant economic and ecological losses, especially if it were to become more widely established in Europe. Early detection tools with high-throughput capacity can increase our readiness to implement mitigation actions against new incursions. This study sought to develop a disease detection method based on volatile organic compound (VOC) emissions to detect F. circinatum on different Pinus species. The complete pipeline applied here, entailing gas chromatography—mass spectrometry of VOCs, automated data analysis and machine learning, distinguished diseased from healthy seedlings of Pinus sylvestris and Pinus radiata. In P. radiata, this distinction was possible even before the seedlings became visibly symptomatic, suggesting the possibility for this method to identify latently infected, yet healthy looking plants. Pinus pinea, which is known to be relatively resistant to F. circinatum, remained asymptomatic and showed no changes in VOCs over 28 days. In a separate analysis of in vitro VOCs collected from different species of Fusarium, we showed that even closely related Fusarium spp. can be readily distinguished based on their VOC profiles. The results further substantiate the potential for volatilomics to be used for early disease detection and diagnostic recognition

BioClay™ prolongs RNA interference-mediated crop protection against Botrytis cinerea

One of the most promising tools for the control of fungal plant diseases is spray-induced gene silencing (SIGS). In SIGS, small interfering RNA (siRNA) or double-stranded RNA (dsRNA) targeting essential or virulence-related pathogen genes are exogenously applied to plants and postharvest products to trigger RNA interference (RNAi) of the targeted genes, inhibiting fungal growth and disease. However, SIGS is limited by the unstable nature of RNA under environmental conditions. The use of layered double hydroxide or clay particles as carriers to deliver biologically active dsRNA, a formulation termed BioClay™, can enhance RNA durability on plants, prolonging its activity against pathogens. Here, we demonstrate that dsRNA delivered as BioClay can prolong protection against Botrytis cinerea, a major plant fungal pathogen, on tomato leaves and fruit and on mature chickpea plants. BioClay increased the protection window from 1 to 3 weeks on tomato leaves and from 5 to 10 days on tomato fruits, when compared with naked dsRNA. In flowering chickpea plants, BioClay provided prolonged protection for up to 4 weeks, covering the critical period of poding, whereas naked dsRNA provided limited protection. This research represents a major step forward for the adoption of SIGS as an eco-friendly alternative to traditional fungicides.Peer reviewe

Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens

Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens

Targeted Delivery of Gene Silencing in Fungi Using Genetically Engineered Bacteria.

Exploiting RNA interference (RNAi) in disease control through non-transformative methods that overcome the hurdle of producing transgenic plants has attracted much attention over the last years. Here, we explored such a method and used non-pathogenic bacteria as a versatile system for delivering RNAi to fungi. Specifically, the RNaseIII-null mutant strain of Escherichia coli HT115(DE3) was transformed with two plasmid vectors that enabled the constitutive or IPTG-inducible production of double-stranded RNAs (dsRNAs) against genes involved in aflatoxins production in Aspergillus flavus (AflC) or virulence of Botrytis cinerea (BcSAS1). To facilitate the release of the dsRNAs, the bacterial cells were further genetically engineered to undergo a bacteriophage endolysin R-mediated autolysis, following a freeze-thaw cycle. Exposure under in vitro conditions of A. flavus or B. cinerea to living bacteria or their whole-cell autolysates induced silencing of AflC and BcSAS1 in a bacteria concentration-dependent manner, and instigated a reduction in aflatoxins production and mycelial growth, respectively. In planta applications of the living bacteria or their crude whole-cell autolysates produced similar results, thus creating a basis for translational research. These results demonstrate that bacteria can produce biologically active dsRNA against target genes in fungi and that bacteria-mediated RNAi can be used to control fungal pathogens

Artificial nanovesicles for dsRNA delivery in spray-induced gene silencing for crop protection

Spray-induced gene silencing (SIGS) is an innovative and eco-friendly technology where topical application of pathogen gene-targeting RNAs to plant material can enable disease control. SIGS applications remain limited because of the instability of RNA, which can be rapidly degraded when exposed to various environmental conditions. Inspired by the natural mechanism of cross-kingdom RNAi through extracellular vesicle trafficking, we describe herein the use of artificial nanovesicles (AVs) for RNA encapsulation and control against the fungal pathogen, Botrytis cinerea. AVs were synthesized using three different cationic lipid formulations, DOTAP + PEG, DOTAP and DODMA, and examined for their ability to protect and deliver double stranded RNA (dsRNA). All three formulations enabled dsRNA delivery and uptake by B. cinerea. Further, encapsulating dsRNA in AVs provided strong protection from nuclease degradation and from removal by leaf washing. This improved stability led to prolonged RNAi-mediated protection against B. cinerea both on pre- and post-harvest plant material using AVs. Specifically, the AVs extended the protection duration conferred by dsRNA to 10 days on tomato and grape fruits and to 21 days on grape leaves. The results of this work demonstrate how AVs can be used as a new nanocarrier to overcome RNA instability in SIGS for crop protection

A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing.

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrusaustraliasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application

A stable antimicrobial peptide with dual functions of treating and preventing citrus Huanglongbing.

Citrus Huanglongbing (HLB), caused by a vector-transmitted phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas), is the most devastating citrus disease worldwide. Currently, there are no effective strategies to prevent infection or to cure HLB-positive trees. Here, using comparative analysis between HLB-sensitive citrus cultivars and HLB-tolerant citrus hybrids and relatives, we identified a novel class of stable antimicrobial peptides (SAMPs). The SAMP from Microcitrus australiasica can rapidly kill Liberibacter crescens (Lcr), a culturable Liberibacter strain, and inhibit infections of CLas and CL. solanacearum in plants. In controlled greenhouse trials, SAMP not only effectively reduced CLas titer and disease symptoms in HLB-positive trees but also induced innate immunity to prevent and inhibit infections. Importantly, unlike antibiotics, SAMP is heat stable, making it better suited for field applications. Spray-applied SAMP was taken up by citrus leaves, stayed stable inside the plants for at least a week, and moved systemically through the vascular system where CLas is located. We further demonstrate that SAMP is most effective on α-proteobacteria and causes rapid cytosol leakage and cell lysis. The α-helix-2 domain of SAMP is sufficient to kill Lcr Future field trials will help determine the efficacy of SAMP in controlling HLB and the ideal mode of application