Foc Gene Research Articles

Identification of SCAR markers linked to the Foc gene governing resistance to Fusarium oxysporum f. sp. cucumerinum in cucumber cv. SMR-18

Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (FOC) is an important soil-borne disease of cucumber in several parts of the world. In this study, we employed amplified fragment length polymorphic (AFLP) markers along with bulked segregant analysis (BSA), in order to identify structural markers linked to the Foc gene that governs resistance to FOC races 1, 2 and 3 in the resistant cv. SMR-18. From the four AFLP markers originally identified at the BSA level, only one (E12M50134 + T) maintained a linkage in coupling phase against the F2 mapping population. AFLP-mediated sequencing was employed in order to ensure the identification and sequencing of the truly linked AFLP fragment. This AFLP marker mapped at a distance of 6.0 cM from the Foc resistance locus and was successfully converted into two sequence-characterized amplified region (SCAR) co-dominant markers. SCAR marker SCE12M50B produced a single fragment linked to Foc and behaved as a dominant marker, while SCE12M50A amplified one strong fragment linked to Foc in parallel with a second faint fragment. The SCE12M50B fragment physically maps on chromosome 2 and lies 1.64 Mb (7,0 cM) away from the microsatellite marker SSR03084, which is closely linked to Ccu locus that controls resistance to scab, caused by Cladosporium cucumerinum in cv. SMR-18.

Evaluation of Biofilm Formation and Frequency of Genes Encoding Curli Fiber, Colanic Acid Capsule and F1c Fimberia Among Uropathogenic Escherichia coli Isolates With Strong Cell Surface Hydrophobicity

Background: The uropathogenic Escherichia coli (UPEC) is an important bacterium that colonizes the bladder mucous membrane as biofilm and causes urinary tract infection (UTI). In addition, UPEC favors long-term persistence and leads to relapses in untreatable UTI. Further, bacterial hydrophobic interactions play a role in bacterial adherence to the surface and facilitate biofilm formation due to adhesion. Similarly, cell surface hydrophobicity (CSH), fimbriae, curli fiber, and colanic capsule allow UPEC isolates to initiate infections. Considering the above-mentioned explanation, this study evaluated the association between genes encoding curli fimbriae, colanic acid (CA) capsule, and f1c fimbriae with biofilm formation and CSH among UPEC isolates. Methods: To this end, 100 Escherichia coli strains were isolated from the urine samples of the patients and were diagnosed by biochemical tests. Furthermore, a tissue culture plate method was used to determine the capacity of biofilm formation, followed by conducting microbial adhesion to hydrocarbons method for CSH determination. Finally, the presence of csgA, csgD, rcsA, rcsC, and foc genes was determined by applying polymerase chain reaction. Results: Totally, 40, 22, and 28 isolates had strong, moderate, and weak biofilm formation capacity, respectively. Moreover, 42 and 38 isolates had strong and moderate CSH. Similarly, among the isolates with strong CSH, 32, 13, and 5 isolates had strong, moderate and weak biofilm formation capacity and the prevalence of csgA, csgD, rcsA, and foc genes was 33, 35, 35, 16, and 29, respectively. Based on the findings, no significant difference was observed between the frequency of csgA, csgD, rcsA, rcsC, and foc genes among the strong, moderate, and weak biofilm producers. Conclusions: In general, there is an association between CSH and the biofilm formation of UPEC isolates. This result showed the role of CSH as an effective factor on bacterial adhesion for the first stage of biofilm formation. However, differentiating the strains is not confirmed regarding their ability to form biofilms and their CSH and the presence of all studied genes.

RNAi-based management for Fusarium wilt of banana

Banana is an important tropical crop and trading commodity in the world. However, Fusarium wilt is a severe risk for the world banana industry. Caused by the soil-borne fungal pathogen, Fusarium oxysporum f. sp. cubense (Foc), the disease is spreading insidiously and has been a source of enormous losses worldwide since the 1800s. Unfortunately, effective control methods are not available yet. The current study used an RNAi strategy, referred to as host-induced gene silencing (HIGS), to confer durable resistance against Fusarium by inhibiting in planta expression of Foc essential genes that are conserved in all Foc races and crucial for fungal virulence. In order to find efficient target genes, a wide search of literature has been conducted and four conserved target regions have been chosen after bioinformatics analysis. Preliminary findings from in vitro fungal inhibition assays showed significant reductions in fungal spore count, germination rate and mycelia growth after treating fungal cultures with double-stranded RNA (dsRNA) targeting four Foc genes, suggesting silencing of these genes was effective to reduce fungal growth. Intron hairpin constructs encoding dsRNA targeting selected Foc genes are in the process of being transformed into Arabidopsis thaliana for proof-of-concept of in planta fungal resistance.

The Foc gene governs resistance to race 3 of Fusarium oxysporum f. sp. cucumerinum in the cucumber cv. SMR-18

The genetics of resistance to race 3 of Fusarium oxysporum f. sp. cucumerinum (FOC race 3) in the cucumber cv. SMR-18 (P1) was investigated by crossing it with the susceptible cucumber cv. Ashley (P2) and evaluating the disease reaction in the parents, F1, F1 reciprocal, F2 and backcross (BC1P1 and BC1P2) populations. Resistance of cv. SMR-18 to FOC race 3 was consistent with that of a single dominant, nuclear gene. To study the linkage relationship between this gene and the Foc gene, which expresses resistance to both FOC races 1 and 2, 73 plants that were backcrossed to the susceptible parent (BC1P2) were self-crossed. Each resulting F2 family was evaluated for resistance or susceptibility to race 1 and race 3 by inoculating different groups of 15–20 plants with each race of the pathogen. Segregation data exhibited an absolute linkage between the Foc gene and the gene controlling resistance to race 3 of the pathogen, since no recombinants were observed and only the two parental classes at a frequency of 50% each were observed. Our data support the concept that the Foc gene, which controls resistance to races 1 and 2, also confers resistance to race 3.

Chickpea-Fusarium oxysporum interaction transcriptome reveals differential modulation of plant defense strategies

Fusarium wilt is one of the major biotic stresses reducing chickpea productivity. The use of wilt-resistant cultivars is the most appropriate means to combat the disease and secure productivity. As a step towards understanding the molecular basis of wilt resistance in chickpea, we investigated the transcriptomes of wilt-susceptible and wilt-resistant cultivars under both Fusarium oxysporum f.sp. ciceri (Foc) challenged and unchallenged conditions. Transcriptome profiling using LongSAGE provided a valuable insight into the molecular interactions between chickpea and Foc, which revealed several known as well as novel genes with differential or unique expression patterns in chickpea contributing to lignification, hormonal homeostasis, plant defense signaling, ROS homeostasis, R-gene mediated defense, etc. Similarly, several Foc genes characteristically required for survival and growth of the pathogen were expressed only in the susceptible cultivar with null expression of most of these genes in the resistant cultivar. This study provides a rich resource for functional characterization of the genes involved in resistance mechanism and their use in breeding for sustainable wilt-resistance. Additionally, it provides pathogen targets facilitating the development of novel control strategies.

Functional analysis of the G-protein α subunits FGA1 and FGA3 in the banana pathogen Fusarium oxysporum f. sp. cubense

The asexual fungus Fusarium oxysporum f. sp. cubense (Foc) is the causal agent of fusarium wilt in bananas (Musa spp.). This fungus poses a threat to banana production throughout the world. Here, two Foc genes, fga1 and fga3, were functionally characterized. These genes encode proteins homologous to the G-protein α subunits GPA1 from Saccharomyces cerevisiae and MAGC from Magnaporthe grisea, respectively. The deletion of fga1 leads to a phenotypic defect in colony morphology and reductions in vegetative growth, conidiation and pathogenicity against the banana plant (Musa spp. cv. Brazil), which was not observed for the Δfga3 deletion mutant. Intriguingly, both Δfga1 and Δfga3 deletion mutants showed declines in intracellular cyclic AMP levels and increases in heat resistance, suggesting that FGA1 regulates growth, development, pathogenicity, and heat resistance, whereas FGA3 modulates heat resistance, potentially through the cAMP-dependent protein kinase A pathway. These findings offer insights into the roles of the G-protein α subunits in the development and pathogenicity of the fungus Foc.

Development of InDel markers linked to Fusarium wilt resistance in cabbage

Cabbage Fusarium wilt (CFW) is a destructive disease causing great losses to cabbage (Brassica oleracea L. var. capitata L.) production worldwide. At present, there are few reports concerning molecular marker research on cabbage resistance to CFW. In this study, 160 double haploid (DH) lines were obtained from the F1 population of a 99–77 (highly resistant to CFW) × 99–91 (highly susceptible to CFW) cross. Insertion–deletion (InDel) markers were designed according to the reference genome sequence of cabbage and the whole-genome re-sequencing data of the two parents. A genetic map of chromosome C06 including seven InDel markers was constructed based on the DH population. Thus, FOC (resistance gene to Fusarium oxysporum f. sp. conglutinans) was located on chromosome C06 and two InDel markers out of the seven, M10 and A1, flanked the gene at 1.2 and 0.6 cM, respectively. Marker A1 revealed a significant consistency with the phenotype assay in the F2 population as well as in 40 inbred lines (96 and 82 %, respectively). This study lays the foundation for fine mapping and cloning of the FOC gene and for marker-assisted selection in cabbage resistance breeding.

An avirulence gene homologue in the tomato wilt fungus Fusarium oxysporum f. sp. lycopersici race 1 functions as a virulence gene in the cabbage yellows fungus F. oxysporum f. sp. conglutinans

Six4, a small protein secreted by Fusarium oxysporum f. sp. lycopersici (Fol) in tomato xylem sap during infection, triggers Fol race 1-specific resistance (I) in tomato. SIX4 is regarded as an avirulence gene. Although SIX4 is considered unique to Fol race 1, we detected this gene in the cabbage yellows fungus F. oxysporum f. sp. conglutinans (Foc) by PCR. Because the genes from Foc and SIX4 in Fol were >99 % identical at the nucleotide level, the Foc gene was designated FocSIX4. The expression of FocSIX4 was detected by RT-PCR in stems and roots of cabbage 8 days after infection with Foc. In contrast with Fol, disruption of FocSIX4 in Foc did not increase virulence to Foc-resistant cabbage cvs. Shutoku-SP and Koikaze. On the contrary, the disruptants had reduced virulence not only on Foc-resistant cultivars but also on Foc-susceptible cv. Shikidori. These results suggested that FocSIX4 is involved in virulence, but not in avirulence, in the cabbage yellows fungus.

Development of chickpea near-isogenic lines for fusarium wilt

Four pairs of near-isogenic lines (NILs) of chickpea with resistance/susceptibility to Fusarium oxysporum f. sp. ciceris (Foc) have been developed in this study. These lines were produced by searching in advanced recombinant inbred lines (RILs) that are segregating for Foc race 5 based on a phenotypic screening. The sequence tagged microsatellite (STMS) marker TA59, closely linked to wilt resistance genes on linkage group 2 (LG2) of the chickpea map, was used to assist the selection of resistant or susceptible genotypes. The NILs were also characterized for disease reaction to Foc races 1A, 2, 3 and 4. Resistance, susceptibility and slow wilting reactions were found in these NILs. Our results suggest that more than one gene controls the resistance to race 5. Combination of the major gene foc-5 linked to TA59 with other gene/s appears to be required to complete resistance, and the absence of these unknown genes leads to slow wilting reactions. The independent differential responses to races 2 and 3 observed in three NILs could be explained as recombination events. This result suggests that foc-2 and foc-3 are delimiting points at opposite ends of a genomic region that includes the remaining foc genes and the TA59 marker. This set of NILs has great potential for studying the genetics and mechanisms of wilt resistance. In addition, the NIL RIP8-94-11 can be used as differential line for Foc race 3; it showed a clear resistance reaction to race 3 and susceptibility to the other Foc races.

Purification, crystallization and preliminary data analysis of FocB, a transcription factor regulating fimbrial adhesin expression in uropathogenic Escherichia coli.

The transcription factor FocB belongs to a family of regulators encoded by several different fimbriae gene clusters in uropathogenic Escherichia coli. Recent findings suggest that FocB-family proteins may form different protein-protein complexes and that they may exert both positive and negative effects on the transcription of fimbriae genes. However, little is known about the actual role and mode of action when these proteins interact with the fimbriae operons. The 109-amino-acid FocB transcription factor from the foc gene cluster in E. coli strain J96 has been cloned, expressed and purified. The His(6)-tagged fusion protein was captured by Ni(2+)-affinity chromatography, cleaved with tobacco etch virus protease and purified by gel filtration. The purified protein is oligomeric, most likely in the form of dimers. NMR analysis guided the crystallization attempts by showing that probable conformational exchange or oligomerization is reduced at temperatures above 293 K and that removal of the highly flexible His(6) tag is advantageous. The protein was crystallized using the hanging-drop vapour-diffusion method at 295 K. A native data set to 2.0 A resolution was collected at 100 K using synchrotron radiation.

Asymptomatic bacteriuria Escherichia coli strain 83972 carries mutations in the foc locus and is unable to express F1C fimbriae

Escherichia coli is the most common organism associated with asymptomatic bacteriuria (ABU). In contrast to uropathogenic E. coli (UPEC), which causes symptomatic urinary tract infection (UTI), very little is known about the mechanisms by which these strains colonize the urinary tract. Bacterial adhesion conferred by specific surface-associated adhesins is normally considered as a prerequisite for colonization of the urinary tract. The prototype ABU E. coli strain 83972 was originally isolated from a girl who had carried it asymptomatically for 3 years. This study characterized the molecular status of one of the primary adhesion factors known to be associated with UTI, namely F1C fimbriae, encoded by the foc gene cluster. F1C fimbriae recognize receptors present in the human kidney and bladder. Expression of the foc genes was found to be up-regulated in human urine. It was also shown that although strain 83972 contains a seemingly intact foc gene cluster, F1C fimbriae are not expressed. Sequencing and genetic complementation revealed that the focD gene, encoding a component of the F1C transport and assembly system, was non-functional, explaining the inability of strain 83972 to express this adhesin. The data imply that E. coli 83972 has lost its ability to express this important colonization factor as a result of host-driven evolution. The ancestor of the strain seems to have been a pyelonephritis strain of phylogenetic group B2. Strain 83972 therefore represents an example of bacterial adaptation from pathogenicity to commensalism through virulence factor loss.

Virulence characteristics of Escherichia coli in acute bacterial prostatitis.

To assess the urovirulence characteristics of Escherichia coli strains causing acute prostatitis, urinary isolates from men with acute prostatitis (n=107) and from women with acute uncomplicated pyelonephritis (n=76) were examined for the prevalence of sfa, foc, and 3 papG allele genotypes and phenotypes and for the production of alpha-hemolysin and cytotoxic necrotizing factor 1. The papG allele III and foc gene were found more frequently and the papG allele II less frequently among prostatitis than from pyelonephritis isolates. A higher proportion of hly+ cnf1+ genotype in prostatitis strains (64% vs. 36%) was particularly striking. Both prostatitis and pyelonephritis strains expressed virulence factors similarly except for a higher proportion of nonhemolytic prostatitis isolates. Although the pathogenetic mechanisms of urinary tract infections in men and women may differ, virulence factors such as adhesins and cytotoxins may have important roles in the pathogenesis of acute prostatitis.

The export systems of type 1 and F1C fimbriae are interchangeable but work in parental pairs

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found, among others, on strains associated with urinary tract infections. Biosynthesis of type 1 and F1C fimbrial organelles requires individual, specialized two-component assembly systems. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structure of the organelles, is very similar; however, the actual sequence homology of the structural elements is not remarkable (34 to 60%). Both gene clusters encode a periplasmically located chaperone and an usher protein, located in the outer membrane, required for organelle biogenesis. Deletion of either element causes abolishment of fimbriation. The present report addresses the question of promiscuity in fimbrial biogenesis. Our data indicate that the two-component export systems of the two organelle systems are reciprocally interchangeable; however, they seem to function only in parental pairs.

Reciprocal exchange of minor components of type 1 and F1C fimbriae results in hybrid organelles with changed receptor specificities

Type 1 and F1C fimbriae are surface organelles of Escherichia coli which mediate receptor-specific binding to different host surfaces. Such fimbriae are found on strains associated with urinary tract infections. The specific receptor binding of the fimbriae is due to the presence of receptor recognition proteins present in the organelles as minor structural elements. The organization of the fim and foc gene clusters encoding these fimbriae, as well as the structures of the organelles, are very similar, although the actual sequence homology of the structural elements is not remarkable; notably, the sequence identity between the minor components of the type 1 and F1C fimbriae is only 34 to 41%. Type 1 fimbriae mediate agglutination of guinea pig erythrocytes, whereas F1C fimbriae do not confer agglutination of any types of erythrocytes tested. However, F1C fimbriae mediate specific adhesion to epithelial cells in the collecting ducts of the human kidney as well as to cells of various cell lines. This report addresses the question of fimbrial promiscuity. Our data indicate that minor fimbrial structural elements can be exchanged between the two fimbrial systems, resulting in hybrid organelles with changed receptor specificity. This is the first study on reciprocal exchange of structural components from two different fimbrial systems.

Nucleotide sequence of the genes coding for minor fimbrial subunits of the F1C fimbriae of Escherichia coli

F1C fimbriae allow uropathogenic Escherichia coli to adhere to specific epithelial surfaces. This adhesive property is probably due to the presence of minor fimbrial components in F1C fimbriae. The foc gene cluster encoding F1C fimbriae has been cloned, as described previously. Here we present the nucleotide sequence (2081 bp) coding for the F1C minor fimbrial subunits. The structural genes code for polypeptides of 175 (FocF), 166 (FocG), and 300 (FocH) amino acids.The deduced amino acids of the F1C minor subunits were compared with the reported sequences of the minor subunits of other types of fimbriae. The data show that the Foc minor subunits are highly homologous to the corresponding Sfa proteins, whereas homology to the minor subunits of type 1 and P fimbriae is much lower.

F1C fimbriae of a uropathogenic Escherichia coli strain: genetic and functional organization of the foc gene cluster and identification of minor subunits

The genetic organization of the foc gene cluster has been studied; six genes involved in the biogenesis of F1C fimbriae were identified. focA encodes the major fimbrial subunit, focC encodes a product that is indispensable for fimbria formation, focG and focH encode minor fimbrial subunits, and focI encodes a protein which shows similarities to the subunit protein FocA. Apart from the FocA major subunits, purified F1C fimbriae contain at least two minor subunits, FocG and FocH. Minor proteins of similar size were observed in purified S fimbriae. Remarkably, some mutations in the foc gene cluster result in an altered fimbrial morphology, i.e., rigid stubs or long, curly fimbriae.