Spores of the PdotA-regulated GFP transformant FJT24 were used to inoculate P. radiata
seedlings to assess GFP expression on needles. The inoculum was checked for GFP expression on the needles for a time period of 2 weeks. In this experiment slight GFP expression was observed on the needle. The GFP expression was not very strong and most spores did not germinate. (Figure 4.17). However, the expression of the PdotA
regulated GFP on needles does suggest early expression of the dothistromin genes on needles.
The experiment was conducted over 14 days. The pine seedlings died after 14 days but no Dothistroma needle blight symptoms were seen, suggesting that the death was not caused by the inoculation with FJT24. A limited time frame for this study and lack of suitable pine seedlings at the time required did not allow the experiment to be repeated.
Figure 4.17: Spores of FJT24 (PdotA::egfp) on pine needles.
GFP expression of FJT24 on pine needles could be observed, although fluorescence was not intense. Only a few spores germinated on the needles (arrows). Bars indicate 50 µm.
4.6 Discussion
D. septosporum expresses EGFP, SGFP and DSRedexpress which makes them useful tools
as reporters in this organism. The transformants with the mammalian codon optimised
egfp under control of the A. nidulans promoter PgpdA showed brighter fluorescence than
to the single transformant obtained with pCT74, which contained the plant codon optimised sgfp under the control of the PtoxA promoter. In some fungi the SGFP does not appear to be successfully expressed (Pöggeler et al. 2003). In this study it was not determined if the lower fluorescence was due to the SGFP itself, to the PtoxA promoter or to the single gene introduction in the only one transformant obtained. For the aim of obtaining GFP reporter strains, the transformants expressing the EGFP were satisfactory.
A parallel study (Barron 2006) highlighted the use of these constitutive GFP reporter strains in the D. septosporum/ pine system as it revealed important information about the disease process and the role of dothistromin as a pathogenicity factor. The constitutive GFP reporter strain FJT21 strain facilitated the observation of the early infection process. Germ tubes of D. septosporum enter the pine needle through stomatal openings (Figure 4.18), but no directed growth towards stomata was observed (Barron 2006) as suggested by Muir and Cobb (2005).
Further the constitutive GFP expressing, dothistromin deficient strain FJT30 (derived from the pksA mutant FJT3) and FJT29 (derived from the dotA mutant FJT2) were successfully used to infect P. radiata seedlings (Figure 4.18, Barron unpublished). GFP fluorescence was detected in fruiting bodies in newly formed lesions (Figure 4.18), suggesting that the infection was caused by FJT30 and not by strains which might have been on the host prior to inoculation with FJT30. Thus, the construction of FJT29 and FJT30 led to the important conclusion that dothistromin is not a pathogenicity factor in the disease.
Figure 4.18: Use of GFP transformants, developed in this study, in pathogenicity trials.
(A, B) FJT21 (wild type; PgpdA::egfp) facilitates the observation of the infection process on needles. Penetration of hyphae through stomata are marked by arrows. (C) Dothistromin deficient strain FJT30 (pksA-; PgpdA::egfp) was also easily observed on needles. Bars in A,B and C indicate 10 µm. (D) Pine needles infected with FJT30 showed disease symptoms. GFP fluorescence of the fruiting bodies identified that the FJT30 caused the disease symptoms and identity of FJT30 in the lesion was subsequently confirmed by PCR. Results shown here are from experiments conducted by Naydene Barron (2006, unpublished).
The successful expression of the egfp gene in D. septosporum allowed the construction of the PdotA regulated egfp reporter strains FJT24 and FJT26. In those strains the GFP was expressed and the gene expression patterns of the native dotA and the
pdotA::egfp appeared to be co-regulated (Figure 4.12). The expression pattern for both dotA
and egfp is also consistent with the dothistromin gene expression observed for the wild type strains in culture (Chapter 3, Figure 4.12). Therefore the expression of the EGFP in those
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The unusual early expression of dothistromin genes (Chapter 3) was also seen in FJT24 and FJT26 colonies, which showed highest GFP fluorescence in younger mycelium. While constitutive GFP expressing strain FJT22 showed ubiquitous GFP fluorescence over the whole colony, dotA-regulated strains FJT24 and FJT26 showed GFP expression predominantly at the growing margin of the colony (Figure 4.8). This is consistent with elevated PdotA-driven gene expression in young hyphae and the early onset of dotA
expression seen in liquid culture (Chapter 3). The same pattern of GFP expression has been observed on both PDA and DM media, suggesting that this is not a media effect, although the expression in DB and PDB media was different in liquid cultures (Section 3.2.3). The detection of GFP in spores and germinating spores of FJT24 and FJT26 (Figures 4.10, 4.11) confirms the early onset of dothistromin gene expression observed in liquid culture as reported in Chapter 3, where dothistromin gene expression was also detected in the spore inocula.
The high level of dothistromin gene expression at an early stage of growth (Chapter 3) also suggests that the regulation of dothistromin biosynthesis is not directly connected with that of sporulation which usually occurs at a late stage of growth. High expression of EGFP in FJT24 and FJT26 was seen in some but not all conidiophores (Figure 4.11). This indicates that some dothistromin synthesis might occur parallel to the sporulation but is not necessarily co-regulated. Interestingly the expression of secondary metabolite genes at an early development stage (depending on the environmental conditions) has been observed in other species and does not appear to be unique in D. septosporum. The pksP gene, encoding a polyketide synthase is involved in both pigment biosynthesis and virulence of A. fumigatus. A GFP-PksP fusion protein was only found in phialides and conidia in vitro indicating a developmentally controlled expression of the gene. But pksP-egfp expression was also detected in hyphae of germinating conidia isolated from the lungs of immunocompromised mice (Langfelder et al. 2001).
The high GFP expression at the margin of the colony supports the hypothesis that dothistromin is involved in the early plant pathogen interaction, where a toxin is secreted in advance of the growing mycelia (Gadgil & Holden 1976, Section 1.2.2). But as the
infection with the dothistromin deficient strains FJT29 and FJT30 showed, the toxin does not appear to be necessary for pathogenicity (Barron 2006). However, GFP expression was also detected in spores of FJT24 on pine needles (Figure 4.17), suggesting the genes are expressed early in planta, even though this experiment was not repeated and GFP fluorescence was weak. As the expression of dothistromin genes was also detected in the spore inocula in Chapter 3, the different levels of GFP fluorescence in the conidiospores might represent different metabolic states, with some spores about to germinate while others are not.
In single hyphae GFP expression in FJT24 and FJT26 was detected in outreaching hyphae in DB cultures but not seen in colony centres (Figure 4.9). However, when grown on cellophane plates of the GFP fluorescence in the dotA-regulated GFP transformants FJT24 and FJT26 was very inconsistent (Figure 4.10), in contrast to the constitutive GFP expressing strains FJT21 and FJT22 (Figure 4.7). While high GFP expression was often seen in germinating spores or small mycelium fragments, other hyphae of similar size or germination status did not show fluorescence. As seen in Figure 4.10F strong fluorescent parts were dispersed between non-fluorescent parts even in single hyphae. This suggests that the loss of GFP fluorescence is not due to a loss of the PdotA::egfp gene. Further, insertion of the PdotA::egfp gene into the FJT24 and FJT26 genome was shown by Southern analyses (Figure 4.4) and two rounds of single spore isolation purified the transformants. This makes it unlikely that the observed mosaic-like GFP expression in FJT24 and FJT26 is caused by the loss of the PdotA::egfp gene. A similar non-uniform pattern of distribution for AF proteins has been seen in areas of colonies in A. parasiticus that are presumably the same age and in a similar environment (Lee et al. 2004). The detection of AF proteins by immunoelectron microscopy showed intensely labelled, weakly labelled, and unlabelled cells adjacent to each other in the same thin layer section. This suggests that the extent and timing of aflatoxin synthesis also varies from cell to cell. Likewise a similar observation was made for other genes in Aspergillus niger. Using GFP reporter genes, it was shown that “exploration hyphae” differentiate, with some
differentiated, even though hyphae in the exploration zone are exposed to the same nutritional conditions (Vinck et al. 2005).
The GFP expression pattern of the PdotA::dotA::egfp transformants FJT31 and FJT32 showed high GFP fluorescence at the margin of colonies as well as in spores and hyphae (Figures 4.13, 4.14). In contrast to FJT24 and FJT26 the GFP was detected more evenly over the colony as well as in older hyphae. This suggests that the protein might be stable and active after translation. This would be a possible explanation for the delayed maximum of toxin production in comparison to the maximum of expression of dothistromin genes seen in Section 3.
Transformants expressing the DotA-EGFP fusion protein also revealed some clues on the intracellular localization of dothistromin biosynthesis. While the EGFP in the D. septosporum transformants appears to be located in the cytosol, the DotA-EGFP fusion protein appeared to be in certain cellular compartments in the fusion strains FJT31 and FJT32. The dappled nature of the DotA-EGFP signal suggests that this fusion protein is localized within cytoplasmic vesicles. While vacuole staining showed some overlapping of GFP signals and vacuolar stain, GFP signals were also observed in unstained vesicles. However, those experiments need to be repeated. On a side note it should be said that the FM4-64 stain used in this study is an endocytosis dependent stain. This is interesting as endocytosis by hyphae of filamentous fungi is still in debate (Fuchs & Steinberg 2005; Read & Kalkman 2003). However, the uptake of FM4-64 and incorporation into vacuoles (Figure 4.16) suggest that endocytosis occurs in D.
septosporum.
Observations of the location of the DotA-EGFP fusion protein has been made near the completion of this study and need to be further investigated. There are several things to be considered. Firstly, the fusion protein is not proven to be functional. Different folding might cause non-functionality and therefore the observed localisation of the protein may be an artefact. Further, the fusion protein has been transformed into the
dotA replacement strain FJT2 and not into the wild type, and the DotA-EGFP fusion
protein did not complement the dotA- strain FJT2. Since morphological changes have been correlated with the production of secondary metabolites the lack of dothistromin, or
Nevertheless, it would be very interesting to see if the synthesis of dothistromin indeed occurs in certain cell organelles. This would imply a regulated mechanism for dothistromin secretion. The identification of such a mechanism might result in new targets for the disease control, if spatial organisation of dothistromin biosynthesis is correlated to auto-protection of the fungi against dothistromin.
The DotA-EGFP fusion protein will facilitate the identification of the organelles of dothistromin sythesis. Although dothistromin antibodies are available (Jones 1993), immunoelectron microscopy to detect the intracellular accumulation of dothistromin is difficult due to technical limitations in the preparation of samples (Jones, personal communication). In contrast, the determination of the intracellular localisation of the DotA-EGFP fusion protein should be more accessible using confocal microscopy. This should allow the identification of organelles required for dothistromin biosynthesis in future work.
The localization of the DotA protein in organelles is supported by the pTARGET analysis, which predicted DotA localization in peroxisomes. Peroxisomes might represent the organelles in which GFP expression is detected in absence of the vacuole stain. Interestingly most of the dothistromin gene products identified so far are predicted to be in cell organelles (Table 4.3). It can be speculated that dothistromin biosynthesis is highly spatially organized and this might play a role in auto-protection. In Cercospora
nicotianae, it was shown that the PDX1 protein is localized in cellular vesicles of
unknown function. PDX1 is thought to have a role in auto-protection against cercosporin, as it is required for growth in the presence of cercosporin and other photosensitizers, and the localization of PDX1 might be important for this (Chung et al. 2002). Because of dothistromin’s toxic properties it might be favourable for D. septosporum if its synthesis is localized in certain cell organelles to keep the intracellular concentration of toxin low. Lee et al. (2002) observed a similar phenotype for the AF protein OmtA in A. parasiticus. OmtA converts ST into o-methylsterigmatocystin and is a protein of the late biosynthesis steps of AF. Cells labeled with OmtA polyclonal antibodies showed patches of
(Nor-1), which are involved in the earlier steps of aflatoxin synthesis are mainly cytoplasmic. Interestingly the AflM protein is the homologue of the DotA protein. But the DotA protein is involved in the late state of dothistromin biosynthesis (Bradshaw et
al. 2002) after the dothistromin and AF pathway are proposed to diverge (Henry &
Townsend 2005, Figure 1.7).
The localization of proteins involved in fungal secondary metabolism in organelles has also been observed for penicillin synthesis in Penicillium chrysogenum (Müller et al. 1991) and localization of prehelminthorospol in organelles was observed in
Bipolaris sorokina (Akesson et al. 1996). The intracellular organization might therefore
in general play an important role in the synthesis of secondary metabolites.
The intracellular localisation of the DotA-EGFP protein might also explain the differences of GFP fluorescence between the PdotA::egfp reporter strains FJT24 and FJT26 and the fluorescence of the PdotA::dotA::egfp transformants FJT30 and FJT31. Vinck et al. (2005) reported that differences in fluorescence using cytoplasmic GFP reporter proteins, and GFP fused to the native protein of interest, were due to cytoplasmic streaming of the GFP in the former case. Similarly the distribution of the cytoplasmic EGFP in FJT24 and FJT26 and the intracellular localization of DotA-EGFP in FJT31 and FJT31 might be responsible for the differences in GFP fluorescence.
In consideration of the similarities of the AF/ST and dothistromin pathways it is very interesting that the dothistromin proteins DotA, DotD, PksA are predicted to be located in peroxisomes. Peroxisomal β–oxidation appears to have an important role for the synthesis of AF/ST, depending on the kind of fatty acids available, and the AF/ST precursor norsolorinic acid was located in peroxisomes (Maggio-Hall et al. 2005). Polyunsaturated fatty acids can be converted to oxylipins. Oxylipins appear to interfere in the perception, metabolism and signalling between host and microbe of each other and are necessary for secondary metabolite production and seed colonisation by Aspergillus species (Tsitsigiannis & Keller 2006; Tsitsigiannis & Keller 2007). If it can be confirmed that the synthesis of dothistromin is located in peroxisomes and therefore connected with the fatty acid metabolism it is feasible that oxylipins play a role as signals or external regulatory molecules, which influence the production of dothistromin, as seen for the
done. The identification of possible signalling molecules for dothistromin production will improve our understanding of the putative roles of dothistromin.
In summary, the construction of D. septosporum GFP reporter strains led to the following results:
• D. septosporum invades P. radiata needles through stomata. The toxin is not
necessary for pathogenicity, although the toxin genes appear to be expressed in
planta.
• The dotA-regulated GFP reporter strains confirmed that the dothistromin genes are highly expressed at an early stage of growth, as seen previously in submerged culture (Section 3). The expression is different in each cell, as also seen for other genes in Aspergillus (Lee et al. 2004; Vinck et al. 2005) although the overall expression of the dothistromin genes appears to decrease with age (Section 3, Figure 4.7).
• The DotA-EGFP protein appears to be at least occasionally located in organelles and the same is predicted for other dothistromin genes. This suggests that the synthesis of dothistromin is spatially organised, as seen for other mycotoxins (Akesson et al. 1996; Lee et al. 2004; Müller et al. 1991).
The early toxin production was supported by the D. septosporum GFP reporter strains, but it was shown that dothistromin is not a pathogenicity factor. Therefore no support was seen for the first hypothesis, implying a role for dothistromin in the early infection process, outlined at the end of Chapter 3.
The second hypothesis, implying a role of dothistromin in competition, is tested in the next chapter.