Nanobodies (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of... more Nanobodies (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb) derived from camelids. Here, we demonstrate production of VHH nanobodies against the SARS-CoV-2 spike proteins in the solanaceous plant Nicotiana benthamiana through transient expression and their subsequent detection verified through western blot. We demonstrate that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2 (ACE2). We present plant production of nanobodies as an economical and scalable alternative to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture.
Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf sc... more Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, Vol. 99, No. 6 (Supplement), 2009 S111 sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. Xa produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in Xa, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. We used Tn5 (transposome) mutagenesis in an attempt to identify additional Xa pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 780 independently derived Tn5 insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculati...
Xanthomonas albilineans (Xa) produces albicidin, a unique and specific toxin that causes foliar s... more Xanthomonas albilineans (Xa) produces albicidin, a unique and specific toxin that causes foliar symptoms of sugarcane leaf scald disease. In X. campestris pv. campestris, a cluster of rpf (for regulation of pathogenicity factors) genes and xanB2 are involved in control of various cellular processes. rpfF and xanB2 encode DSF (diffusible signal factor) and DF (diffusible factor), respectively, which are two quorum sensing signalling molecules. Both quorum sensing systems appear to be used by Xa, since mutation of rpfF in Florida strain XaFL07-1 resulted in reduced protease production, and mutation of xanB2 resulted in loss of xanthomonadin (yellow pigment) production. Mutations of rpfF and xanB2 were verified by PCR analyses. Mutations of rpfF and complementation in trans were also verified by use of an X. campestris DSF reporter strain. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, exhibited pencil line symptoms indicative of albicidin production on emerging le...
Xanthomonas albilineans (Xa) est une bacterie qui se multiplie dans le xyleme et qui est responsa... more Xanthomonas albilineans (Xa) est une bacterie qui se multiplie dans le xyleme et qui est responsable de l'echaudure des feuilles de la canne a sucre. Les symptomes de la maladie varient d'une tres fine ligne blanche foliaire a la necrose et au fletrissement complet des feuilles infectees, conduisant ainsi a la mort de la plante. Xa produit la toxine albicidine qui bloque la differenciation des chloroplastes, ce qui aboutit a l'apparition de symptomes foliaires. L'albicidine etait le seul facteur de pathogenie connu jusqu'a recemment, meme si des souches non productrices d'albicidine etaient toujours capables de coloniser la canne a sucre. Des mutants d'insertion aleatoire (Tn5) de la souche de Xa FL07-1 originaire de Floride ont ete produits afin d'identifier de nouveaux facteurs de pathogenie. Mille mutants ont ete inocules au cultivar de canne a sucre CP80-1743, moderement sensible a l'echaudure des feuilles. Les symptomes ont ete releves sur le...
Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. ... more Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. X albilineans produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in X albilineans, yet albicidin-deficient mutant strains are still able to efficiently colon\ze sugarcane. We used TnS (transposome) mutagenesis in an attempt to identifY additional X albilineans pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 1,216 independently derived TnS insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculation. In ad...
The genome of Xanthomonas albilineans, the causal agent of sugarcane leaf scald, contains a clust... more The genome of Xanthomonas albilineans, the causal agent of sugarcane leaf scald, contains a cluster of genes called rpf for regulation of pathogenicity factors. This cluster of genes encodes a quorum sensing (also called cell-cell signaling) system that controls several cellular and biological processes in Xylella fastidiosa, Stenotrophomonas maltophilia and several species of Xanthomonas. The rpf gene cluster is especially used by these pathogenic bacteria to regulate the expression of factors contributing to virulence such as extracellular polysaccharides, extracellular enzymes and flagellum synthesis. This cell-cell signaling is mediated by a signal molecule called DSF (for Diffusible Signaling Factor), and gene rpfF has been shown to be required for production of DSF. Perception of DSF and signal transduction depends on a two-component signal transduction system which comprises the sensor kinase RpfC and the regulator RpfG. The role of these rpf genes in X albilineans is not yet...
Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured pla... more Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including ‘Candidatus L. asiaticus’ (CLas) and ‘Ca. L. solanacearum’ (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely re...
Huanglongbing is causing economic devastation to the citrus industry in Florida, and threatens th... more Huanglongbing is causing economic devastation to the citrus industry in Florida, and threatens the industry everywhere the bacterial pathogens in the Candidatus Liberibacter genus and their insect vectors are found. Bacteria in the genus cannot be cultured and no durable strategy is available for growers to control plant infection or pathogen transmission. However, scientists and grape growers were once in a comparable situation after the emergence of Pierce’s disease, which is caused by Xylella fastidiosa and spread by its hemipteran insect vector. Proactive quarantine and vector control measures coupled with interdisciplinary data-driven science established control of this devastating disease and pushed the frontiers of knowledge in the plant pathology and vector biology fields. Our review highlights the successful strategies used to understand and control X. fastidiosa and their potential applicability to the liberibacters associated with citrus greening, with a focus on the inte...
Xylella fastidiosa infects a wide range of plant hosts and causes Pierce’s disease (PD) of grapev... more Xylella fastidiosa infects a wide range of plant hosts and causes Pierce’s disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naph...
Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium &q... more Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium "Candidatus Liberibacter asiaticus" and transmitted by Asian citrus psyllids (Diaphorina citri). Two prophage genomes, SC1 and SC2, integrated in "Ca. Liberibacter asiaticus" strain UF506 were described previously, and very similar prophages are found resident in the majority of "Ca. Liberibacter asiaticus" strains described worldwide. The SC1 lytic cycle is marked by upregulation of prophage late genes, including a functional holin (SC1_gp110); these late genes are activated when "Ca. Liberibacter asiaticus" is in planta, but not when infecting the psyllid host. We previously reported that the holin promoter is strongly and constitutively active in Liberibacter crescens (a cultured proxy for uncultured "Ca. Liberibacter asiaticus") but is suppressed in a dose-dependent manner by crude aqueous extracts from D. citri applied exogenously. Her...
Molecular plant-microbe interactions : MPMI, Jan 27, 2015
Candidatus Liberibacter asiaticus (Las) is transmitted by psyllids and causes Huanglongbing (HLB)... more Candidatus Liberibacter asiaticus (Las) is transmitted by psyllids and causes Huanglongbing (HLB), a lethal disease of citrus. Most pathogenic Las strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high copy excision plasmid encoding a reactive oxygen species (ROS) scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus, and suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into E. coli and L. crescens (Lcr), a culturable proxy for Las. Transformed Lcr cells showed 20-25% enhanced resistance to H2O2 on agar plates, 47% higher enzymatic activity and enhanced growth in liquid cultures. A non-classical secretion potential was predicted for SC2_gp095 and secretion from Lcr was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta result...
Nanobodies (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of... more Nanobodies (VHH antibodies), are small peptides that represent the antigen binding domain, VHH of unique single domain antibodies (heavy chain only antibodies, HcAb) derived from camelids. Here, we demonstrate production of VHH nanobodies against the SARS-CoV-2 spike proteins in the solanaceous plant Nicotiana benthamiana through transient expression and their subsequent detection verified through western blot. We demonstrate that these nanobodies competitively inhibit binding between the SARS-CoV-2 spike protein receptor binding domain and its human receptor protein, angiotensin converting enzyme 2 (ACE2). We present plant production of nanobodies as an economical and scalable alternative to rapidly respond to therapeutic needs for emerging pathogens in human medicine and agriculture.
Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf sc... more Xanthomonas albilineans (Xa) is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, Vol. 99, No. 6 (Supplement), 2009 S111 sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. Xa produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in Xa, yet albicidin-deficient mutant strains are still able to efficiently colonize sugarcane. We used Tn5 (transposome) mutagenesis in an attempt to identify additional Xa pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 780 independently derived Tn5 insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculati...
Xanthomonas albilineans (Xa) produces albicidin, a unique and specific toxin that causes foliar s... more Xanthomonas albilineans (Xa) produces albicidin, a unique and specific toxin that causes foliar symptoms of sugarcane leaf scald disease. In X. campestris pv. campestris, a cluster of rpf (for regulation of pathogenicity factors) genes and xanB2 are involved in control of various cellular processes. rpfF and xanB2 encode DSF (diffusible signal factor) and DF (diffusible factor), respectively, which are two quorum sensing signalling molecules. Both quorum sensing systems appear to be used by Xa, since mutation of rpfF in Florida strain XaFL07-1 resulted in reduced protease production, and mutation of xanB2 resulted in loss of xanthomonadin (yellow pigment) production. Mutations of rpfF and xanB2 were verified by PCR analyses. Mutations of rpfF and complementation in trans were also verified by use of an X. campestris DSF reporter strain. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, exhibited pencil line symptoms indicative of albicidin production on emerging le...
Xanthomonas albilineans (Xa) est une bacterie qui se multiplie dans le xyleme et qui est responsa... more Xanthomonas albilineans (Xa) est une bacterie qui se multiplie dans le xyleme et qui est responsable de l'echaudure des feuilles de la canne a sucre. Les symptomes de la maladie varient d'une tres fine ligne blanche foliaire a la necrose et au fletrissement complet des feuilles infectees, conduisant ainsi a la mort de la plante. Xa produit la toxine albicidine qui bloque la differenciation des chloroplastes, ce qui aboutit a l'apparition de symptomes foliaires. L'albicidine etait le seul facteur de pathogenie connu jusqu'a recemment, meme si des souches non productrices d'albicidine etaient toujours capables de coloniser la canne a sucre. Des mutants d'insertion aleatoire (Tn5) de la souche de Xa FL07-1 originaire de Floride ont ete produits afin d'identifier de nouveaux facteurs de pathogenie. Mille mutants ont ete inocules au cultivar de canne a sucre CP80-1743, moderement sensible a l'echaudure des feuilles. Les symptomes ont ete releves sur le...
Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. ... more Xanthomonas albilineans is a systemic, xylem-invading pathogen that causes sugarcane leaf scald. Leaf symptoms vary from a single, white, narrow, sharply defined stripe to complete wilting and necrosis of infected leaves, leading to plant death. X albilineans produces the toxin albicidin that blocks chloroplast differentiation, resulting in disease symptoms. Albicidin is the only previously known pathogenicity factor in X albilineans, yet albicidin-deficient mutant strains are still able to efficiently colon\ze sugarcane. We used TnS (transposome) mutagenesis in an attempt to identifY additional X albilineans pathogenicity factors. Sugarcane cultivar CP80-1743, moderately susceptible to leaf scald, was inoculated by the decapitation method with 1,216 independently derived TnS insertions in Florida strain XaFL07-1. Leaf scald symptoms were recorded on emerging leaves one month after inoculation, and stalk colonization by the pathogen was determined two months after inoculation. In ad...
The genome of Xanthomonas albilineans, the causal agent of sugarcane leaf scald, contains a clust... more The genome of Xanthomonas albilineans, the causal agent of sugarcane leaf scald, contains a cluster of genes called rpf for regulation of pathogenicity factors. This cluster of genes encodes a quorum sensing (also called cell-cell signaling) system that controls several cellular and biological processes in Xylella fastidiosa, Stenotrophomonas maltophilia and several species of Xanthomonas. The rpf gene cluster is especially used by these pathogenic bacteria to regulate the expression of factors contributing to virulence such as extracellular polysaccharides, extracellular enzymes and flagellum synthesis. This cell-cell signaling is mediated by a signal molecule called DSF (for Diffusible Signaling Factor), and gene rpfF has been shown to be required for production of DSF. Perception of DSF and signal transduction depends on a two-component signal transduction system which comprises the sensor kinase RpfC and the regulator RpfG. The role of these rpf genes in X albilineans is not yet...
Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured pla... more Axenically cultured Liberibacter crescens (Lcr) is a closely related surrogate for uncultured plant pathogenic species of the genus Liberibacter, including ‘Candidatus L. asiaticus’ (CLas) and ‘Ca. L. solanacearum’ (CLso). All Liberibacters encode a completely conserved gene repertoire for both flagella and Tad (Tight Adherence) pili and all are missing genes critical for nucleotide biosynthesis. Both flagellar swimming and Tad pilus-mediated twitching motility in Lcr were demonstrated for the first time. A role for Tad pili in the uptake of extracellular dsDNA for food in Liberibacters was suspected because both twitching and DNA uptake are impossible without repetitive pilus extension and retraction, and no genes encoding other pilus assemblages or mechanisms for DNA uptake were predicted to be even partially present in any of the 35 fully sequenced Liberibacter genomes. Insertional mutations of the Lcr Tad pilus genes cpaA, cpaB, cpaE, cpaF and tadC all displayed such severely re...
Huanglongbing is causing economic devastation to the citrus industry in Florida, and threatens th... more Huanglongbing is causing economic devastation to the citrus industry in Florida, and threatens the industry everywhere the bacterial pathogens in the Candidatus Liberibacter genus and their insect vectors are found. Bacteria in the genus cannot be cultured and no durable strategy is available for growers to control plant infection or pathogen transmission. However, scientists and grape growers were once in a comparable situation after the emergence of Pierce’s disease, which is caused by Xylella fastidiosa and spread by its hemipteran insect vector. Proactive quarantine and vector control measures coupled with interdisciplinary data-driven science established control of this devastating disease and pushed the frontiers of knowledge in the plant pathology and vector biology fields. Our review highlights the successful strategies used to understand and control X. fastidiosa and their potential applicability to the liberibacters associated with citrus greening, with a focus on the inte...
Xylella fastidiosa infects a wide range of plant hosts and causes Pierce’s disease (PD) of grapev... more Xylella fastidiosa infects a wide range of plant hosts and causes Pierce’s disease (PD) of grapevines. The type 1 multidrug resistance (MDR) efflux system is essential for pathogenicity and survival of bacterial pathogens in planta. X. fastidiosa, with a single MDR system, is significantly more vulnerable to inhibition by small-molecule treatments than most bacterial pathogens that typically carry redundant MDR systems. A high-throughput cell viability assay using a green fluorescent protein-marked strain of X. fastidiosa Temecula 1 was developed to screen two Prestwick combinatorial small-molecule libraries of drugs and phytochemicals (1,600 chemicals in total) approved by the Food and Drug Administration and European Medicines Agency for cell growth inhibition. The screens revealed 215 chemicals that inhibited bacterial growth by >50% at 50 µM concentrations. Seven chemicals proved to lyse X. fastidiosa cells at 25 µM, including four phytochemicals. Menadione (2-methyl-1,4-naph...
Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium &q... more Huanglongbing (HLB) is a severe disease of citrus caused by an uncultured alphaproteobacterium "Candidatus Liberibacter asiaticus" and transmitted by Asian citrus psyllids (Diaphorina citri). Two prophage genomes, SC1 and SC2, integrated in "Ca. Liberibacter asiaticus" strain UF506 were described previously, and very similar prophages are found resident in the majority of "Ca. Liberibacter asiaticus" strains described worldwide. The SC1 lytic cycle is marked by upregulation of prophage late genes, including a functional holin (SC1_gp110); these late genes are activated when "Ca. Liberibacter asiaticus" is in planta, but not when infecting the psyllid host. We previously reported that the holin promoter is strongly and constitutively active in Liberibacter crescens (a cultured proxy for uncultured "Ca. Liberibacter asiaticus") but is suppressed in a dose-dependent manner by crude aqueous extracts from D. citri applied exogenously. Her...
Molecular plant-microbe interactions : MPMI, Jan 27, 2015
Candidatus Liberibacter asiaticus (Las) is transmitted by psyllids and causes Huanglongbing (HLB)... more Candidatus Liberibacter asiaticus (Las) is transmitted by psyllids and causes Huanglongbing (HLB), a lethal disease of citrus. Most pathogenic Las strains carry two nearly identical prophages similar to SC1 and SC2 in strain UF506. SC2 was observed to replicate as a moderately high copy excision plasmid encoding a reactive oxygen species (ROS) scavenging peroxidase (SC2_gp095), a predicted lysogenic conversion factor. SC2_gp095 was expressed at significantly higher levels in periwinkle than in citrus, and suppressed in psyllids. SC2_gp095 was cloned in a shuttle vector and transformed into E. coli and L. crescens (Lcr), a culturable proxy for Las. Transformed Lcr cells showed 20-25% enhanced resistance to H2O2 on agar plates, 47% higher enzymatic activity and enhanced growth in liquid cultures. A non-classical secretion potential was predicted for SC2_gp095 and secretion from Lcr was confirmed by enzymatic and Western blot analyses. Transient expression of SC2_gp095 in planta result...
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