Figure 1.
M. oryzae show tolerance towards copper stress.
A) Prolonged effects of copper stress on fungal growth and phenotype was studied by growing M. oryzae cells in presence or absence of copper for 7 days. A regime of copper concentrations was used as shown in a-e which represents 0, 0.1, 1.0, 2.5 and 5.0 mM copper concentrations. Fungal cells showed dense fungal growth and change in hyphal color from gray to white with increasing copper concentration. Dense aerial hyphae were observed in presence of 2.5 mM copper (f). B) Effect of copper on M. oryzae growth and its uptake was determined. To estimate, copper acquisition and its effect on fungal growth, 4 days old M. oryzae cultures were exposed to copper (0–5 mM) for 18 h. Percent growth inhibitory rate was calculated by comparing growth of stress exposed samples with control (untreated) sample. Inhibitory rate and copper acquisition by M. oryzae showed positive correlation (r = 0.97).
Table 1.
Details of transposable elements (TEs) and oligonucleotides used in the study.
Figure 2.
Stress induced genetic variations based on repetitive DNA elements.
Mutation rates for transposable elements (TEs) were compared with simple sequence repeats (SSR) and TE/SSR regions. Mutation rates were calculated based on number of altered DNA bands generated in stress exposed samples as compared to control. Gray and white bars represent mutation rates determined for heat shock and copper stress exposed samples respectively. All the dataset are analyzed with two-way analysis of variance (ANOVA) with Bonferroni post-tests. Results were found extremely significant at p<0.0001. Data are presented as means ± standard error mean (SEM).
Figure 3.
Stress induces genomic instability and genetic diversity in M. oryzae.
A) Genomic template stability (GTS) index of M. oryzae transposable elements upon stress exposure. GTS index was calculated by analyzing mutant bands generated in stress exposed samples as compared to control sample. Data represents the means for stress exposed samples and was analyzed with one-way analysis of variance (ANOVA) with Dunnett's multiple comparison test (each group compared to the control). Data are presented as means of GTS index of stress exposed samples. Results showed Pyret with lowest GTS index, suggesting Pyret as the most unstable transposable element (TE) upon stress induction. B) High genetic diversity was observed among M. oryzae isolates. Genotypic profiles were obtained from 23 M. oryzae isolates from diverse geographical regions of India and Japan using PyR1 outward primer (Table 1) derived from LTR-retrotransposon Pyret. Results suggest bursts of Pyret under field conditions. C) Correlation between TE copy number (Y-axis) and mutation rate (X-axis) was determined. Pot2 with high copy number showed no genetic variations upon stress induction, whereas for Pyret linear correlation was observed between its copy number and mutation rate.
Table 2.
Magnaporthe oryzae isolates used for genetic variability studies.
Figure 4.
Genetic variability among M. oryzae isolates.
DNA profiles were obtained for M. oryzae isolates using outward primers PyR1 (upper) and Pot2L2 (lower) derived from LTR-retrotransposon Pyret and DNA transposon Pot2 respectively. Details of PyR1 and Pot2L2 primers are provided in table 1. Lanes 1 to 9 represents M. oryzae isolates from similar agro-climatic region of India (Table 3). Lane M represents Fermentas GeneRuler 100 bp Plus DNA Ladder. As compared to Pot2, Pyret generated high intra-regional genetic variations among M. oryzae isolate.
Table 3.
Magnaporthe oryzae isolates used for intra-regional genetic variability studies.
Figure 5.
Differential effect of stress types on mutation rates of transposable elements.
A) Genomic DNA templates from M. oryzae fungal cultures exposed to regime of durations and doses of heat shock (HS) and copper (Cu) respectively were analyzed for induced genetic variations. GTS index was determined for different doses of copper stress (left panel) and durations of heat shock (right panel). Data sets are significant at p<0.001 compared to control as analysed by two-way ANOVA with Bonferroni post-tests. B) Mutation rate data for different TEs upon exposure to copper (white) and heat shock (gray). DNA transposon Pot3 showed high mutation rates for heat shock samples. For copper stress, highest mutation rate was observed for LTR-retrotransposon Pyret and MINE. Data sets are the means of mutation rate. Data sets are analysed by two-way ANOVA with Bonferroni post-tests. ***denotes extremely significant results, ** highly significant results at p<0.001, ns non- significant at p<0.001.
Figure 6.
Genomic template stability (GTS) index of M. oryzae transposable elements upon stress exposure.
GTS index of transposable elements were calculated based on the number of mutant bands obtained in M. oryzae samples exposed to regime of heat shock (HS 1-3 h) and copper (Cu 0.1, 1.0 and 2.0 mM) stress. For each TE, GTS index (Y-axis) for M. oryzae stress exposed samples (X-axis) is determined. Data are presented as means from 3 independent experiments. Panels A to H represents GTS index of TEs Pyret, MINE, Mg-SINE, MGLR3, MAGGY, Pot3, Grasshopper (Grh) and Pot2 respectively. Data sets are significant compared to control as analysed by two-way ANOVA with Bonferroni multiple comparison post-tests. Overall, LTR-retrotransposons Pyret (A) and MAGGY (E) showed lowest genomic stability upon stress induction. Pyret showed slightly lower GTS for copper concentrations tested, as compared to heat shock (A). For MAGGY, GTS was not affected by stress type/dose/duration except for HS-3h sample (E). For copper stress, LTR-retrotransposon MINE showed lowest GTS (B). LTR-retrotransposons MGLR3 (D) and Grasshopper (G) showed high GTS index (76 and above) for all stress exposed samples except for HS-3h (52.9). Similarly for all copper doses, Mg-SINE showed similar GTS index (71.4 and above), whereas low GTS of 57.1 and 42.9 for HS-1h and HS-2h samples respectively (C). DNA transposon Pot3 (F) showed more sensitivity towards heat shock, whereas Pot2 (H) showed 100% genomic stability upon stress exposition. Results suggested genome template stability differs for type/family of TEs upon stress exposition.
Table 4.
Summary of the cloning and sequence analyses of representative stress altered DNA bands.
Figure 7.
Relative contributions of the transposable elements (TEs) in stress induced DNA variations in M. oryzae.
Pie chart depicts estimated relative contributions of the transposable elements (TEs) attributable to stress induced DNA variations in M. oryzae genome. The listed TEs include members of class I (MAGGY, Pyret, Grasshopper, MGLR3, MINE and Mg-SINE) and II (Pot3 and Pot2) families. Estimate is based on the mutation rate data calculated using mutant bands generated in stress exposed sample as compared to control. LTR-retrotransposons Pyret and MAGGY accounted for 40% of the observed genetic variations upon stress induction. The remaining 60% variations are attributed to Pot3, MINE, Mg-SINE, Grh and MGLR3.
Figure 8.
Model for TEs role as stress capacitors to promote genomic rearrangements in fungal pathogen.
In the presence of appropriate stress, cells experience genomic shock that generates signals (stochastic or programmed) to induce transpositional activity. This leads to the insertion of transposable elements within regulatory and coding regions of genes resulting in genomic rearrangements.