The deactivation of the Gα subunit Gna1
from S. nodorum
has proven fruitful to further understanding the pathogenesis of this fungal pathogen [9
]. The lack of sporulation and reduced pathogenicity of the resulting gna1
strain sparked further investigation into the molecular and phenotypic attributes of this mutant strain largely because a determination of the molecular processes underpinning the phenotype could lead to more targeted control of the pathogen. Subsequent analysis of the gna1 strain to identify downstream-regulated targets and processes has uncovered many interesting aspects of the disease including mycotoxin production.
Based on the success of dissecting the gna1
phenotype, we have extended this study to include the analysis of the Gβ and Gγ subunits of the heterotrimeric G-protein through a reverse genetics approach. Strains of S. nodorum
lacking these genes displayed variety of independent phenotypes during growth in vitro
. One of the most apparent phenotypic defects under normal growth conditions was the complete lack of pycnidia formation or accompanying asexual sporulation. This phenotype is shared by other S. nodorum
strains possessing defects in signalling pathways, and as such, was consistent with earlier findings in S. nodorum
]. Along with growth defects in vitro
, the mutant strains also exhibited different abilities to cause disease. Lesion formation on leaves inoculated with strains lacking with Gna1
was delayed but appeared comparable to that of the wild type after two weeks post inoculation. Leaves inoculated with Gba1
though failed to elicit any response from the leaves after 5 dpi, and only a very mild chlorotic response was evident after two weeks. This implies that Gba1
has a critical role in disease development in S. nodorum
. Given the almost complete lack of symptom development, it could be suggested that Gba1
, like StuA
], has a role in effector regulation. However this is only speculation and requires further analysis.
Nutrient sensing in the S. nodorum gna1, gba1 and gga1 strains
Dramatic growth differences between the mutant strains and the wild-type SN15 were noted on agar plate medium. On V8PDA, SN15 grows radially symmetrical with pycnidia forming in distinct circadian bands [15
]. The gna1
mutant strains both show a similar banding pattern, in mycelial growth, indicating that these strains have not lost the capacity to perceive a light signal.
The radial growth of all three mutant strains 10 dpi was reduced by comparison to SN15 on all tested media. The variation in radial growth of the mutant strains when growing on different carbon sources confirmed that the S. nodorum G-protein(s) play(s) a role in carbon source utilization. In comparison to the wild-type SN15, which displayed a statistically similar radial growth rate when provided with arabinose, fructose, glucose, sucrose or trehalose as a sole carbon source.
The comparatively slower growth of gna
1 on sucrose was interesting when considering this strain’s slower growth on glucose, but significantly higher growth on fructose. Kraakman et al., (1999) showed that the GPCR Gpr1 binds extracellular glucose in the yeast Saccharomyces cerevisiae
and stimulates cAMP synthesis through the Gα subunit Gpa2. Likewise Lemaire et al., (2004) showed both glucose and sucrose induced cAMP signalling through the receptor Gpr1, however it was not fructose-induced. Although deletion of either Gpr1
did not result in a reduced growth rate in S. cerevisiae
, the strains in the study were not limited to a single carbon source [16
]. However, an altered capacity to sense and respond to the presence of these sugars will inevitably affect growth. The reduced radial growth rate of the S. nodorum gna1
mutant when solely provided with glucose or sucrose compared to fructose therefore could be due to a reduced capacity for sensing glucose and sucrose and imply similar functions for the S. nodorum
Gna1 and yeast Gpa2. It has also been shown that in binding glucose, the GPCR Gpr1 will fail to cause the normal rapid activation of adenylate cyclase if the glucose is not internalized and phosphorylated [16
], which may further explain slower growth in response to glucose in strains where the deactivated subunit causes a lesser response to glucose.
Irrespective of the speculated extracellular sensing roles of these G-protein subunits, the difference in growth rates across S. nodorum gna1, gba1 and gga1 strains when provided with these carbon sources can be explained by processes biochemically downstream. Alterations in catabolic processes may have arisen as a result of the mutations. The growth rates of gna1 on each of fructose and glucose, compared to sucrose, for example is consistent with processes downstream of sucrose (α-D—fructofuranosyl α-D-glucopyranoside) hydrolysis, which yields one unit of fructose and one of glucose. Given that gna1 grows faster on fructose, it suggests that glucose may be feeding less efficiently into glycolysis in this strain.
Interestingly the seemingly inherently slower growth rate of S. nodorum gga1
under most conditions is comparable with each of the mutant strains when provided with trehalose as a sole carbon source. The radial growth rates on trehalose could also implicate all three subunits in processes downstream of extracellular sensing. The hydrolysis of trehalose (α-D-glucopyranosyl-α-D-glucopyranoside) yields two glucose units, yet the growth of gba
1 is particularly slower on trehalose than glucose, which may suggest rather than a glycolytic inefficiency as mentioned above, a reduced capacity to hydrolyse trehalose, or even a diminished capacity to sense the signals that would otherwise cue the cell to catabolise trehalose. Changes to trehalose metabolism have been shown to have dramatic effects on sugar metabolism in general, and shown to have severe implications for phytopathogenicity [17
], so the reduced capacity to use trehalose as a sole carbon source has likely had direct implications on fungal fitness.
Metabolite secretion by the S. nodorum gna1, gba1 and gga1 strains
S. nodorum gna1
has been shown to secrete brown pigments comprised of tyrosine, phenylalanine and L-DOPA into the growth medium, first observed in the discolouration of the growth medium [9
]. Discolouration at the growth medium is also an attribute of S. nodorum
SN15 and the gba1
mutant strains. The carbon source dependency and intensity of discolouration of the medium also imply implications at least for primary metabolism, in the mutant strains. The carbon-source dependency of media discolouration identifies differences in metabolite secretion resulting from likely metabolic changes. These could potentially result from the inefficient use of metabolites or products of metabolism due to blockages or even over-active biochemical pathways. Together with the reduced growth rates on different media, the Gna1
mutations appear to have introduced metabolic inefficiencies.
In the later observed cultures of S. nodorum gna1, gba1 and gga1, where pycnidia formation was studied, more intense secretions could be seen. It’s likely that the intensity of media discolouration was heightened by accumulation over the extended culture period however it may also be that the secretions changed as the cultures’ phenotypes changed. It’s also possible that the increased concentration of secreted metabolites in the culture medium played a role in triggering the formation of pycnidia in these strains. Either way, the increased presence of secreted metabolites in these strains whilst undergoing pycnidial differentiation adds further interest to the identity of these secreted metabolites.
Pathogenicity and asexual sporulation of the S. nodorum gna1, gba1 and gga1 strains
The capacity to rapidly increase fungal inoculum density by releasing spores from pycnidia following infection of the wheat plant by S. nodorum
is fundamental to the success and consequently the impact of SNB. S. nodorum gna1
were all unable to sporulate during infection of the wheat leaf, however although this defect may slow disease amplification, sporulation is clearly not a prerequisite for leaf necrosis. The inability for disease caused by infection with the gba1
strain to progress beyond chlorosis however, may implicate necrotrophic effector production in S. nodorum
as positively regulated by G-protein signalling through the Gβ subunit Gba1 [14
]. It is interesting to note that the requirement of the Gβ and Gγ subunits for infection in different fungal plant pathogens varies. For example, it has been previously demonstrated that GBB1 in Gibberella monoliformis
is not required for pathogenicity whist the orthologous protein in the related Fusarium oxysporum
]. Our data clearly show that gene encoding for the Gβ subunit, Gba1
, is required for S. nodorum
to cause disease on wheat.
Whilst sporulation was not observed for the gna1, gba1 or gga1 strains in planta, the observations of asexual sporulation described in vitro are of considerable interest. The capacity for the gna1, gba1 and gga1 strains to develop pycnidia during prolonged incubation at 4°C from an already matured, yet non-sporulating culture adds further interest and potential for using these strains to dissect these fundamental processes in S. nodorum.
The physical characteristics of the mutant pycnidia observed in vitro were also of interest. In S. nodorum SN15, differentiation of cells forming the ostiole of the mature pycnidial wall was observed, but was not seen for the mutant pycnidia. It is likely that spores could not be released by the fungal strains because of this defect. Cirrus containing the spores was also observed in SN15, but not in the mutant pycnidia. Without the cirrus, it is unlikely there would be enough turgor pressure to release the spores, even with the formation of a wild-type ostiole, and it may be that this pressure plays a role in the formation of the ostiole in the S. nodorum pycnidium.
The pycnidia of the strains gga1 and gba1 are comparatively misshapen and less mature in appearance than those of SN15 and gna1. However, because these strains do develop viable spores, they may not actually be less mature, but perhaps this manifestation is a consequence of these two strains lacking the capacity to develop such a well-defined pycnidial wall.
In conclusion, this study has demonstrated the critical, and yet independent, roles of the heterotrimeric G-protein subunits in S. nodorum. Each of these subunits was found to play a role in in vitro and in planta growth, albeit with varied roles. As had been previously observed for the gna1 strain, gba1 and gga1 strains were unable to sporulate when grown under normal growth conditions. However, prolonged incubation of these strains at 4°C appeared to complement the sporulation defect and pycnidia, containing viable pycnidiospores, were differentiated in each of the mutants. The mechanism of how colder temperatures induce sporulation in these mutants is clearly of interest and is the focus of ongoing studies. It should be noted that whilst single event homologous recombination events were demonstrated for each of the mutants generated in this study, future studies will attempt to complement these strains to provide unequivocal proof of the role of these in the above described phenotypes.