Expression of the ftf1 and ftf2 genes from the pathogenic fungus Fusarium oxysporum (Expresión de Los Genes Ftf1 Y Ftf2 Del Hongo Patógeno Fusarium Oxysporum).
Creators: name: De Vega Bartol, JJ
Publisher: name: Zenodo
Year: 2009
Subjects: thesis

description: <strong><em>De Vega Bartol, JJ. Expression of the ftf1 and ftf2 genes from the pathogenic fungus Fusarium oxysporum (Expresión de Los Genes Ftf1 Y Ftf2 Del Hongo Patógeno Fusarium Oxysporum). Doctoral dissertation, University of Salamanca, Salamanca, Spain. 16/02009.</em></strong> <strong>OBJECTIVES</strong> This thesis expects to continue the study of the ftf1 and ftf2 transcription factors in the pathogenic fungus F. oxysporum. In order to accomplish this task, we aimed at the following specific objectives: a) characterization of virulence in new isolates of F. oxysporum f.sp. phaseoli, b) analysis of ftf1 and ftf2 expression, c) new insights in the genomic architecture of the copies of both genes in the genus Fusarium and other plant pathogens, d) characterization of transformants with ftf1 and ftf2 altered expression, and e) identification of genes regulated by ftf1 and ftf2 as a way to know about the function of both genes. <strong>BACKGROUND</strong> Our group has isolated F. oxysporum in common bean (P. vulgaris) and runner bean (P. coccineous) fields in the region of El Barco de Ávila (Ávila, Spain). The comparison of these Spanish isolates with pathogenic F. oxysporum f.sp. phaseoli isolates from other geographic origin allowed the identification of three new VCG called 0166, 0167 and 0168 (Alves-Santos et al., 1999), which together constitute a new race of the pathogen. These pathogenic isolates can be divided in two groups, highly virulent and weakly virulent, on the basis of the virulence differences as determined by using the CIAT scale (Pastor- Corrales &amp; Abawi, 1987) in inoculation assays performed on spanish varieties of common bean: (Alves-Santos et al., 2002a). In a previous study (Alves-Santos et al., 2002b), we isolated a sequence characterized amplified region (SCAR) marker, named SCAR-B310A280, that enables the in planta detection of highly virulent F. oxysporum f.sp. phaseoli strains as early as 5 hours post-infection. This SCAR marker contains an ORF with a Zn(II)2Cys6 binuclear cluster DNA-binding domain and a medium homology region or domain (MHR), both domains are fungal specific and characteristic of transcription factors. The gene was called ftf1 (Fusarium transcription factor 1) (Ramos et al., 2007), and it can be found not only in forma especialis phaseoli, but also in thirteen different formae speciales such as betae, pisi, lycopersici, cubense, etc. (Nuñez-Corcuera, 2004; Ramos et al., 2007). Expression of the gene ftf1 could not be detected in mycelium growing in liquid media neither by Northern hybridizations nor RT-PCR (Reverse transcription PCR). However, in planta transcription of ftf1 is increased about 24 hours after plant inoculation, as detected by Real-time RT-PCR. This result suggests that ftf1 has a role in the establishment of the fungus within the plant and/or the progress of the disease (Ramos et al. 2007). Multiple copies of ftf1 are present in highly virulent strains of F. oxysporum f.sp. phaseoli, as it could be demonstrated by Southern hybridizations carried out with probes from the ftf1 coding region (M18B-M19A) and the ftf1 promoter (B310-A280) Gene disruption experiments showed that two or more of the copies are transcriptionally active. (Ramos et al. 2007). These Southern hybridizations also showed a faint hybridization that can also be found in DNA from weakly virulent and non-pathogenic strains. A new gene highly homologous (83%) to the ftf1 coding region was isolated and designated ftf2 (Fusarium transcription factor 2). By contrast, ftf1 and ftf2 promoter homology is only about 65%, which suggests that there may be a different regulation of both genes. Also, ftf2 homologues were found in a wide range of filamentous fungi (Ramos, 2005). <strong>RESULTS</strong> Virulence of twenty-five F . oxysporum strains isolated from P . coccineous was characterized by greenhouse inoculation assays performed on common bean and runner bean plants. Inoculation of runner bean plants, showed that nine isolates display a new virulence phenotype characterized by CIAT scale values close to 9 two weeks after infection. They were designated supervirulent strains. Nine isolates presented a lower virulence phenotype than the previous group, with CIAT scale values over 7 after four weeks, similar results to those displayed by the common bean highly virulent group. The third group showed CIAT scale ratings below 7 four weeks after infection. Similar values than showed in common bean by the weakly virulent group, so they were designated weakly virulent too. CIAT scale ratings did not increase when inoculates plants were allowed to grow for up to eight weeks. The statistical significance of F. oxysporum f.sp. phaseoli virulence groups in runner bean was verified by D.P.R. (Disease Progression Rate) quantification and an Analysis of variance (One-way ANOVA). Supervirulent isolates had a D.P.R. over 8, highly virulent isolates had a D.P.R. between 8 and 7, and weakly virulent isolates had a D.P.R. below 6.5. Also, virulence of strains isolated in runner beans was tested in inoculations assays on common bean plants. In this case there was no difference between the supervirulent and the highly virulent group. Four new vegetative compatibility groups (VCG) were found among the F. oxysporum strains isolated from runner bean plants. VCG 0169 includes most of the isolates, VCG 01610 includes two isolates and VCGs 01611 and 01612 are composed by only one isolate each. All isolates have the same sexual idiomorph or mating-type MAT1-2. RFLP analysis of the ribosomal DNA intergenic spacer (IGS) showed two polymorphic patterns previously described as IGS-A haplotype and IGS-B2 haplotype. (Alves-Santos et al., 1999). There is no correlation between virulence groups, VCGs and IGS haplotypes. The number of copies of the ftf1 gene present in the genome of isolates characterized in these work was analyzed by Southern hybridization using probes obtained from the ftf1 promoter and the 5’ coding region. Supervirulent strains showed one more band than the highly virulent, indicating the presence of another polymorphic copy of the ftf1 gene. However, some isolates classified in infection essays in runner bean like highly virulent, and even an isolated classified like weakly virulent in runner bean, also showed this additional band. All strains isolated from runner bean have several copies of the ftf1 gene, confirming that this gene is exclusive of highly aggressive (highly virulent and supervirulent) strains. The expression of gene ftf2 was quantified in a representative strain of each described virulence group, RNA samples were obtained from mycelium grown in liquid medium (PDB) and from inoculated common bean plants. The expression pattern of ftf2 along time is slightly different for each strain analyzed, but the differences do not account for clear up or down regulation. Either expression was relatively stable since the earlier sampling times or increased until a stable expression pattern. No correlation of expression patterns with strain virulence could be found. The expression pattern of gene ftf2 during the infection of common bean plants was similar to that found during growth in liquid cultures: quite stable with a small increase in time and without differences among strains with different virulence. This stability in the gene expression is characteristic of constitutive genes involved in fundamental functions. Also, the lack of up or down regulation in supervirulent or highly virulent strains suggests the lack of a role for ftf2 in virulence. The expression of gene ftf1 was measured in the supervirulent strains characterized in this work, and results were compared with those formerly obtained in highly virulent strains (Ramos, 2005; Ramos et al., 2007). The expression of ftf1 during axenic growth in liquid culture is mainly invariable along time and similar in a supervirulent strain and a highly virulent strain. Previous studies have shown a dramatic increase in ftf1 expression 24 hours after infection in common bean plants inoculated with highly virulent strains (Ramos et al., 2007). In this work, using another Real-time PCR measuring procedure, we have detected a 15-fold increase for the supervirulent strain (SP13) and a 11-fold increase for the highly virulent strain (SP1). The difference may suggest a functional relationship between the number of ftf1 functional copies, strain virulence and ftf1 expression maximum. The dramatic increase in the ftf1 expression during infection but not during axenic growth strongly supports a relevant role for ftf1 in the early infection stages. The search for ftf2 homologues reveal unique sequences in the genomes of both pathogenic fungi (as Magnaporthe grise and Aspergillus nidulans) and non-pathogenic fungi (as Neurospora crassa and Podospora anserina). This distribution suggests gene ftf2 is not involved in pathogenicity. There is a ftf2 homologous sequence in the genomes of F. oxysporum, F. graminearum and F. verticilloides, and the ftf2 flanking regions are conserved in the three species. When regions of the sequence that are highly homologous between ftf1 and ftf2 are used as probes in Blast searches against the genome sequences of Fusarium oxysporum, Fusarium verticillioides and Fusarium graminearum, a single sequence can be detected in the last two genomes, and this is ftf2. On the contrary, the sequenced genome of F. oxysporum f.sp. lycopersici strain 4287 reveals up to eleven sequences homologous to the F. oxysporum f.sp. phaseoli genes ftf1 and ftf2. The coding region is conserved among these eleven sequences (homology around 90%) but the promoter region shows important differences because of the presence of the transposon Impala into the promoter of five of these ftf1 homologous sequences. Also, in these five copies the last aminoacids of the protein have been lost. These facts raise questions about the functionality of these copies. We have used Southern hybridizations to analyze the number of ftf1 polymorphic copies in different F. oxysporum formae speciales. The number of ftf1 copies is highly variable among formae speciales and even among isolates of the same forma specialis. In f.sp. lycopersici, we have found isolates showing 4 or 5 ftf1 copies (as happened in f.sp. phaseoli). In general, the number of polymorphic copies is lower than the number of copies found in the genome analysis. Also, a fragment from the beginning of the ftf2 coding region was amplified from DNA samples of representative strains of different F. oxysporum formae especiales. Isolates from different formae especiales showed sequences mainly similar. For example, among f.sp. phaseoli and lycopersici isolates; or among betae, nicotiana and pini isolates. The sequenced f.sp. phaseoli ftf1 copy (ftf1a) is more similar to one of the f.sp. lycopersici ftf1 copies than this last to the rest of copies of the same strain. The fact that several copies of gene ftf1 are functional rules out the use of gene knock-out procedures to obtain null mutants. In order to generate transformants with an attenuated or suppressed ftf1 and ftf2 expression, we designed a silencing construction (pJJWA) base on a binary plasmid in which two copies of the sequence amplified from a completely conserved region in ftf1 and ftf2, but arranged in opposite senses, have been introduced. To facilitate the loop formation a spacer, typically an intron, is usually included between the two sequences. Our silencing construction used a region of the target gene, only present in one of the amplicons, as spacer, with efficient results. Because of the high homology of the target region in genes ftf1 and ftf2, we expected to silence both genes at the same time. Transformants were obtained from four wild-type strains representing the different virulence groups: supervirulent (SP13), highly virulent (SP1) and weakly virulent (SP4) strains, and also from a non-pathogenic strain (AB82). Transformants carrying the silencing construction cloned in plasmid pJJWA showed a reduction in the growth rate, rate of sporulation and, in some cases, also in the virulence. However, when we measured the ftf2 expression in the silenced transformants by Real-time PCR we did not always observe a reduction; on the contrary, some transformants showed increased levels of some regions of the transcript. Our hypothesis is that this is the consequence of an amplification of the silencing in a similar way than had been described in plants: Aberrant RNAs may be the template to produce new dsRNA, that later would generate secondary siRNA. The reduction in the growth rate observed in axenic growth cultures (PDA) is observed in F. oxysporum f.sp. phaseoli and f.sp. lycopersici transformants. To rule out that the decreased growth rate was a consequence of the transformation process, we performed transformations with the construction pS3, that contains the hygromycin resistance cassette (the hph gene under the control of the TrpC promoter and the Trp terminator from Aspergillus nidulans), but does not harbour the silencing target regions genes of the ftf1 and ftf2 genes. In this case, no growth alterations were observed. In F. oxysporum f.sp. phaseoli, the growth rate reduction was observed in transformants obtained from the four wild-type strains. Among transformants obtained with the same construction from the same wild type, the growth alterations display a gradient. Although they may be separated into two groups according to ANOVA tests. Some of the transformants showed clear significant alterations (strongly silenced transformants). Growth rates were compared when some of the silenced transformants were cultured in media containing different carbon and nitrogen sources, and three different pH. We observed differences after 3 days of growth that disappeared after 7 days. There were differences in the growth rates between the wild-type and silenced transformants strains in medium without glucose source, medium without nitrogen source and acid medium (pH4), which suggest that silenced transformants are more sensible to nutrients starvation stress. Also, sporulation rates were quantified after 3 and 7 days. Some transformants had an appreciable and significant reduction while other, though they displayed growth rate reduction, showed non significantly different sporulation rates, slightly smaller than the wild-type strains. To verify whether ftf2 silencing had effects in virulence, we carried out inoculation assays to compare the infection produced by weakly virulent strains FOP-SP4 (which do not have any ftf1 copy) with that produced by the corresponding silenced transformants. The CIAT scale ratings and D.P.R values were similar for all the strains, further supporting the hypothesis that ftf2 has no role on virulence. The effect of ftf1 + ftf2 gene silencing was also analyzed in inoculation assays of common bean plants with a supervirulent strain (FOP-SP13), a highly virulent strain (FOP-SP1) and a sample of the corresponding silenced transformants. Six FOP-SP13-derived transformants that showed a significant reduction in growth rate were compared to FOP-SP13. Two of them (1.05 and 1.07), showed CIAT ratings similar to FOP-SP13 (CIAT ratings over 7 three weeks after inoculation). Two of them (1.06 and 1.09), showed CIAT ratings below to those induced by FOP- SP13 (CIAT ratings under 6 three weeks after inoculation). Finally, the last two analyzed transformants (1.04 and 1.11) showed disease ratings clearly lower than those exhibited by FOP- SP13 (CIAT ratings under 4 three weeks after inoculation). Four transformants obtained from the highly virulent strain FOP-SP1, that displayed a clear growth reduction, were also tested. Three of them (2.06, 2.07 and 2.20) did not show differences in the CIAT scale ratings with the wild-type strain FOP-SP1. They all showed CIAT scale values over 7 three weeks after inoculation. However, he fourth transformant (2.08) showed a clear reduction in virulence, with CIAT ratings under 5 three weeks after inoculation. The D.P.R. values were also lower than those obtained for the wild types, supporting the evidence of a virulence reduction in transformants 1.06, 1.09, 1.04, 1.11 and 2.04, that would be induced by the silencing of gene ftf1. To investigate the possible function of the genes ftf1 and ftf2, we decided to identify genes regulated by ftf2. Using subtractive hybridization (SSH) we compared the RNAs transcribed in strain FOP-SP4 (a weakly virulent strain that only harbours the ftf2 gene) and the silenced transformant 3.01, in mycelia growth in axenic culture for 24 hours. We obtained 46 sequences out of 60 clones, from which, 18 were different ORFs. Among them, we selected 12 sequences, after a Blast search with the F. oxysporum genome, to undergo an expression study by Real-time PCR. To this purpose relative expressions of the 12 ORFs were compared in mycelia of the wild- type strain FOP-SP4 and the transformant 3.01 grown for 48 hours, and also in mycelia of the strain FOP-SP4 and the transformant 3.15 grown for 24 hours. All the putative genes supposedly regulated by ftf2 showed lower expression in the silenced transformants than in FOP-SP4. It is worth to note the finding of genes, among those supposedly regulated by ftf2, involved in stress response, mitochondrial hypoxia and response to nutritional stress. Also, a Glutamine amidotransferase class-I coding ORF and a Piruvate descarboxilase with a tiamine binding domain (tiamine is a cofactor in the carbon pathway) coding ORF were found. As well, an Aspartyl protease highly homologous to T. harzianum SA76 CWDE was found. Finally, we found a Cyclin and two proteins without known conserved domains. One of the unknown ORFs is the most repeated sequence in the library. <strong>DISCUSSION AND CONCLUSIONS</strong> In conclusion, we have characterized a new virulence group of isolates in F. oxysporum f.sp. phaseoli. These strains isolated from P. coccineous plants that showed symptoms of Fusarium wilt, have been characterized as supervirulent strains, in order to distinguish them from the previously described highly virulent and weakly virulent strains. All the supervirulent strains harbour an additional copy of the gene ftf1. However, some highly virulent and weakly virulent isolates also show this extra copy. Gene expression analysis carried out in bean plants inoculated with supervirulent strains show a higher upregulation of ftf1 than that observed when inoculations were performed with highly virulent strains, which suggests that the extra ftf1 copy is functional. Gene ftf2 is a transcription factor with an activator role, as all the putative genes identified in the substractive hybridization experiment showed a smaller expression in the ftf2 silenced transformants than in the weakly virulent wild-type strain FOP-SP4. Expression of gene ftf2 is mainly constant in time, both in mycelium grown in axenic cultures or in inoculated plants. This is a characteristic expression pattern of constitutive genes. Gene ftf2 seems to be important for regular growth and sporulation, because silenced transformants show a significant reduction of the growth and sporulation rates. Some evidences seem to indicate that ftf2 could be involved in metabolism adaptation to nutrient stress conditions, because the higher growth differences between silenced transformants and the wild- type strains were found when they were grown in media with no carbon or nitrogen sources at shorter times. Also, two of the ORFs supposed
description: Summary in English; Thesis in Spanish.