Identification of MoALR2 and MoMNR2

We identified CorA Magnesium transporters from the M. oryzae genome (http://www.broadinstitute.org/annotation/genome/magnaporthe_grisea/MultiHome.html) by a BLAST_P search using the full length S. cerevisiaeAlr1proteinsequence (859 amino acids). We obtained two putative orthologs in the M. oryzae genome: Mgg_08843 (47% identity) and Mgg_09884 (49% identity), which are named MoAlr2 and MoMnr2 respectively. Both these proteins have two transmembrane domains towards the carboxy terminus, which are followed by conserved residues of (W/F) GMN, and hence belong to the CorA superfamily of Mg²⁺ transporters. MoAlr2 is a 622 amino acid protein with a CorA domain spanning amino acids 310–617 (Pfam). Strongly preferred model (ExPASy) for MoAlr2 predicts that the protein has two transmembrane helices (565–582; in-out) and (596–615; out-in), with the N-terminus facing the cytosol; the protein has been predicted to be localized to the plasma membrane (WoLFPSORT). MoMnr2is a 814 amino acid protein with a CorA domain spanning amino acids 491–809 (Pfam). MoMnr2 is predicted to have two transmembrane helices (756–773; in-out) and (788–806; out-in), with the N- terminus facing the cytosol. In S. cerevisiae, the ScMnr2 protein has been shown to be localized to vacuolar membrane. The MoAlr2 and MoMnr2 CorA domains have ~ 48% identity with that of the S. cerevisiae CorA domain. Multiple sequence alignment of CorA superfamily transporters across different species including yeast and several filamentous pathogenic and non-pathogenic fungi was done using full length protein sequence and the phylogenetic relationship of these CorA proteins is presented (Fig 1A). The CorA transporters included in the analysis form two clades. The MNR clade has W (tryptophan) just before the conserved sequence motif of GMN, while the ALR clade has F (phenylalanine) prior to the GMN sequence (Fig 1B).

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Fig 1. Identification of MoALR2 and MoMNR2.

(A)The tree was constructed using the Neighbor-Joining method based on alignment of full length sequences of CorA proteins of Magnaporthe oryzae (XP_003713862, XP_003709977), Saccharomyces cerevisiae (EDV10489, EDV09793, YKL064W), Aspergillus fumigatus (XP_754049), Aspergillus nidulans (CBF70700, CBF77902), Candida tropicalis (XP_002548119), Schizosaccharmoyces pombe (NP_595545) and Neurospora crassa (NCU11312, NCU03312). The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. All positions containing gaps and missing data were eliminated. Evolutionary analyses were done using MEGA5. (B) Amino acid sequence alignment of CorA proteins Magnaporthe oryzae (XP_003713862, XP_003709977), Saccharomyces cerevisiae (EDV10489, EDV09793, and YKL064W), Aspergillus fumigatus (XP_754049), Aspergillus nidulans (CBF70700, CBF77902), Candida tropicalis (XP_002548119), Schizosaccharmoyces pombe (NP_595545) and Neurospora crassa (NCU11312, NCU03312) was performed using Clustal Omega. CorA consensus sequence motifs (Y/FGMN) for the above proteins are highlighted. (C) Conidia were harvested and treated with polyclonal antibodies raised against the CorA domain of MoMnr2. TRITC labeled secondary antibodies were used for staining. Oregon green 488 staining was used to visualize the vacuole. (D) S. cerevisiaeΔalr1Δalr2 mutant (CM66) was transformed with pYES2-MoALR2, pYES2-MoMNR2 and pYES2-MoMNR2_489-812. The transformants were grown overnight on SD+Gal+leu+lys-ura+500mM Mg²⁺ and different dilutions were spotted on SD+Gal–ura+leu+lys containing 4mM Mg²⁺ and 500mM Mg²⁺ and grown at 28°C for 4 days. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g001

CorA transporters are known to be present on the plasma membrane as well as on organelles to form a system of Mg²⁺ uptake and compartmentalization that maintains cytoplasmic Mg²⁺ homeostasis. In yeast, ScAlr2 has been shown to be a plasma membrane protein while ScMnr2 is a vacuolar membrane protein. The subcellular localization of CorA transporters in M. oryzae was done by indirect immunolocalization using polyclonal antibodies against the MoMnr2 CorA domain. In wild type, the CorA transporters localized to plasma membrane and vacuole (Fig 1C). Vacuolar localization was seen by co-localization with Oregon Green 488 staining (which stains vacuolar lumen). In the MoMNR2 knockout (Δmnr2), the MoAlr2 protein was found to be restricted to the plasma membrane (Fig 1C).

To confirm the nature of these Mg²⁺ transporters functionally, complementation with the M. oryzae genes was carried out in yeast. The S. cerevisiae Δalr1Δalr2 mutant CM66 [23] is a haploid disruptant for both ALR1 and ALR2 genes. Unlike the wild type, the double mutant is unable to grow at 4mM Mg²⁺, indicating a defect in Mg²⁺ uptake. To test the ability of MoALR2 and MoMNR2 to complement the Mg²⁺ uptake defect in the double mutant CM66, transformants over-expressing either MoALR2 or MoMNR2, were first grown in SD media containing 500mM Mg²⁺ and then different dilutions were spotted on SD media containing 4mM Mg²⁺. The transformants were able to grow even at 4mM Mg²⁺ like the wild type, while the mutant could not (Fig 1D), suggesting that both MoALR2 and MoMNR2 could rescue the Mg²⁺ uptake defect and hence have a role in Mg²⁺ transport. Further, a truncated MoMNR2 was also able to complement the function in the yeast mutant, suggesting that amino acids of the CorA domain at the Carboxyl terminus (489–812 amino acids) are sufficient for Mg²⁺ transport.

CorA Mg²⁺ transporters alter metal ion composition in M. oryzae

Targeted disruption of MoALR2 and MoMNR2 through homologous recombination was attempted to investigate the functions of CorA Mg²⁺ transporters in M. oryzae. MoMNR2 knockouts (Δmnr2) were generated using a Zeocin resistance cassette in the wild type strain B157 (WT). Three independent transformants were confirmed by Southern blotting for targeted gene deletion (S1B Fig). Immunostaining of Δmnr2 with CorA antibodies showed staining only of plasma membrane due to MoAlr2, while vacuolar staining was absent (Fig 1C). In contrast, only non-homologous (ectopic) transformants were obtained for MoALR2 despite multiple attempts using different conditions of selection (S1 Table). Knockout of MoALR2 was also attempted in the Δku80 background known to aid homologous integration. But screening of all the transformants resulted only in ectopic integrants (S1 Table). Thus, we speculate that MoALR2 is essential for viability of M. oryzae.

To establish the role of MoALR2 by an alternative approach, the gene was silenced in both WT and Δmnr2 backgrounds using a stretch of 110bp complementary to the 5’ UTR of MoALR2 [24], cloned in the vector pSD2. The knockdown was validated by analysis of relative expression of MoALR2 and MoMNR2 by quantitative Real Time PCR (qRT-PCR) in the transformants. Transformants in the WT background showed transcript levels of MoALR2 ranging from 48% to 85%, while those in the background of Δmnr2 showed transcript levels ranging from 66% to 88% compared to WT (S2 Table). No transcripts of MoMNR2 were detected in Δmnr2 background transformants, while the levels of MoMNR2 did not change in the MoALR2 knockdown transformants in wild type background, thereby confirming the specificity of the cassette used for MoALR2 silencing. Since MoALR2 could not be silenced more strongly in the Δmnr2 background, to obtain transformants with further reduced transcript levels of MoALR2, an alternative knockdown approach for simultaneous silencing of both MoALR2 and MoMNR2 was also used. As these two genes show high similarity in the CorA domain, we carried out simultaneous silencing using an antisense construct targeted to this region. The knockdown was validated by qRT-PCR of MoALR2 and MoMNR2 in the transformants. The transformants showed transcript levels ranging from 30% to 80% and 37% to 90% for MoALR2 and MoMNR2 respectively, compared to WT (S2 Table).

To be sure that reduced transcript levels translated into decline in transporter levels, we used a Co(III) Hexaammine sensitivity test. Unlike WT, growth of CorA knockout/knockdown strains is not sensitive to cation hexaammines, demonstrating that the inhibition is mediated by an interaction between CorA and the hexaammines [25]. We tested the sensitivity of knockdown transformants as compared to WT. Two independent knockdown transformants from each category, namely Alr2 silencing (WT+siALR2), Δmnr2+siALR2 and simultaneously silenced for MoALR2 and MoMNR2, which were least sensitive to Co(III)Hex., were selected for further study. Southern blot analysis confirmed single site integration in these knockdown transformants (S2A Fig). The degree of resistance to Co(III)Hex. of the selected transformants, Δmnr2+siALR2_79, Δmnr2+siALR2_66, WT+siALR2_56, WT+siALR2_48, A2 and A15 (Fig 2A) correlated with the degree of silencing of MoALR2. The expression levels (%) of MoALR2 and MoMNR2 in the transformants relative to WT were Δmnr2 (126, 0), Δmnr2+siALR2_79 (79, 0), Δmnr2+siALR2_66 (66, 0), WT+siALR2_56 (56, 90), WT+siALR2_48 (48, 89), A2 (43, 52) and A15 (30, 37) (Fig 2B). Interestingly, the Δmnr2 knockout was more sensitive to Co(III)Hex. than wild type, possibly due to up-regulation of MoALR2 transcripts observed in the three independent Δmnr2 knockouts. Failure of Δmnr2 to show Co(III)Hex. resistance, once again suggests its intracellular localization, since cation hexaammines are known to affect only surface transporters, as seen with MoALR2 knockdowns.

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Fig 2. CorA Mg²⁺ transporters affect metal ion composition in M. oryzae.

(A) CorA specific inhibitor (Cobalt (III) hexaammine (Co (III) Hex.) was added to YEG medium at concentrations of 300μM and 400μM. Sensitivity was assessed relative to Wild type (WT) five days post inoculation and growth was measured for WT, Δmnr2 and knockdown transformants. (B) mRNA levels of MoALR2 and MoMNR2 were estimated by qRT-PCR. Transcript levels were normalized to that of WT. (C) Intracellular levels of Mg²⁺ in the knockout and knockdown transformants were estimated by XRF. The values are expressed as percentage values, with 100 corresponding to WT at 4mM Mg²⁺. (D) Intracellular levels of Mg²⁺ in WT were estimated in presence of 4mM extracellular Mg²⁺ and EDTA at 8hrs. The values are expressed as percentage values, with 100 corresponding to 4mM Mg²⁺. ** means P value at <0.0001 and * means significant at P value <0.05. Values are the mean of two independent experiments with each performed in triplicates.

https://doi.org/10.1371/journal.pone.0159244.g002

Western blot analysis of selected transformants was done to determine whether protein levels were also reduced in them. The sequence of the CorA domain from MoMnr2 has 50% identity with that of MoAlr2, so the antibodies raised against the CorA domain detected both MoAlr2 (70 kDa) and MoMnr2 (90 kDa). The transformants studied showed lower protein levels of these CorA Mg²⁺ transporters than wild type B157 (S2B Fig).

We next investigated whether a decrease in Mg²⁺ transporter levels affects the intracellular levels of metal ions (mainly Mg²⁺ and Ca²⁺), using X-ray Fluorescence Analysis (XRF) of hyphae obtained under standard growth conditions (medium containing 4mM Mg²⁺). We found a decrease in the intracellular levels of Mg²⁺ in the knockdown transformants (Fig 2C) while intracellular levels of Ca²⁺ did not change significantly (data not shown). Significant decreases in Mg²⁺ levels were seen when the silencing of MoALR2 was >50%. For instance, XRF analysis of A2 and A15 showed that Mg²⁺ levels were reduced to 25% and 21% of WT levels respectively. Thus we show that CorA Mg²⁺ transporters play a significant role in maintenance of intracellular metal ion composition. Next, to look at the effect of extracellular Mg²⁺ availability, we determined the intracellular Mg²⁺ levels in presence of extracellular EDTA (a Mg²⁺ chelator). The intracellular levels of Mg²⁺ in WT decreased in presence of EDTA (Fig 2D) indicating a need for extracellular Mg²⁺ and its uptake for maintenance of the intracellular ionic milieu.

To investigate whether extracellular Mg²⁺ supplementation restores intracellular Mg²⁺, the two knockdown transformants which showed maximum silencing of MoALR2, A2 and A15, were grown in presence of higher concentrations of extracellular Mg²⁺ (50mM and 250mM). When supplemented with 50mM Mg²⁺, the intracellular Mg²⁺levels in A2 and A15 increased to 62% and 42% (from 25% and 21%) respectively (Fig 3A) and rose further at 250mM Mg²⁺ (Fig 3B). This could be either due to enhanced uptake by the CorA transporters in the knockdown transformants, or due to non-specific transport at higher levels of Mg²⁺ by other metal ion transporters. In WT, while intracellular Mg²⁺ levels remained unchanged at 50mM extracellular Mg²⁺, there was a drastic increase at 250mM Mg²⁺ (Fig 3A and 3B). The increased Ca²⁺/Mg²⁺ ratio observed in A2 and A15 at 4mM Mg²⁺, also returned to lower levels in presence of 50mM Mg²⁺ (Fig 3C).

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Fig 3. XRF analysis of knockdown transformants.

(A), (B) Intracellular levels of Mg²⁺ at 4mM, 50mM and 250mM extracellular Mg²⁺ in WT and the double knockdown transformants, A2 and A15. The values are expressed as percentages, with 100 corresponding to the WT at 4mM Mg²⁺. (C) Ratios of Ca²⁺ to Mg²⁺ at two different concentrations of Mg²⁺ in WT, A2 and A15. Values are the mean of two independent experiments with each performed in triplicates. Error bar denote SD. ** means P value at <0.0001 and * means significant at P value <0.05.

https://doi.org/10.1371/journal.pone.0159244.g003

Mg²⁺ dependent expression of MoALR2 and MoMNR2

The expression profile of MoALR2 and MoMNR2 was studied in WT grown in presence of EDTA, 50mM and 250mM extracellular Mg²⁺ for different lengths of time (2 hours and 6 hours). The addition of EDTA resulted in up-regulation of both MoALR2 and MoMNR2. MoALR2 showed a biphasic mode of regulation with respect to different concentrations of Mg²⁺; transcript levels decreased at 50mM Mg²⁺ both at 2 hours and 6 hours, while at 250mM Mg²⁺, the transcript level increased both at 2 hours and 6 hours (Fig 4A). The transcript levels of MoMNR2 decreased with increasing concentrations of Mg²⁺ (Fig 4B). To examine how the levels of MoAlr2 and MoMnr2 proteins change with extracellular Mg²⁺ levels, Western blot analysis was also done. The levels of both proteins increased in presence of EDTA. In the presence of 50mM Mg²⁺ the level of MoAlr2 was comparable to that seen with 4mM Mg²⁺ alone (i.e. no EDTA), both at 2 hours and 6 hours, but increased at 250mM Mg²⁺ both at 2 hours and 6 hours compared to that at 4mm Mg²⁺ (Fig 4C). The level of MoMnr2 protein showed the same trend as seen at transcript level, decreasing with increasing concentration of Mg²⁺ and with increasing time interval (Fig 4D).

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Fig 4. Regulation of MoALR2 and MoMNR2at mRNA and protein level with respect to extracellular Mg²⁺.

(A) mRNA levels of MoALR2 were estimated in WT by qRT-PCR at different concentrations of extracellular Mg²⁺. (B) mRNA levels of MoMNR2 were estimated by qRT-PCR at different concentrations of extracellular Mg²⁺. Transcript levels were expressed as relative values, with 1 corresponding to levels at 4mM. (C) Western blot analysis of WT for MoAlr2 at different concentrations of extracellular Mg²⁺. (D) Western blot analysis for MoMnr2 at different concentrations of extracellular Mg²⁺. ** means P value at <0.0001 and * means significant at P value <0.05. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g004

Ca²⁺ dependent expression of MoALR2 and MoMNR2

Calcium (Ca²⁺) is a natural antagonist of Mg²⁺ [23, 9, 14]. To evaluate how expression of the plasma membrane Mg²⁺ transporter MoALR2 changes with increasing concentration of Ca²⁺, transcript and protein levels were studied in WT. The transcript level of MoALR2 increased at 50mM and 250mM extracellular Ca²⁺ compared to control (where extracellular Ca²⁺ was chelated with EGTA) (Fig 5A). Protein level of MoAlr2 was higher than in the EGTA-treated control, but MoMnr2 protein level remained constant even at high concentrations of Ca²⁺ (Fig 5B). It is likely that high intracellular concentration of Ca²⁺ induced increased expression of MoALR2 as part of a feedback mechanism to maintain a favorable Ca²⁺/Mg²⁺ ratio.

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Fig 5. Regulation of MoALR2 in WT at mRNA and protein level with respect to extracellular Ca²⁺.

(A) mRNA levels of MoALR2 were estimated in WT by qRT-PCR at different concentrations of extracellular Ca²⁺. The transcript levels were expressed as relative values, with 1 corresponding to levels at 4mM. Error bar denote SD. (B) Western blot analysis for MoAlr2 at different concentrations of extracellular Ca²⁺. ** means P value at <0.0001 and * means significant at P value <0.05. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g005

Altered Cation sensitivity in knockdown transformants

Mg²⁺ is the most abundant divalent cation in the cell. A change in Mg²⁺ levels affects metal ion homeostasis and may alter sensitivity to heavy metal ions. We assayed the sensitivity of Δmnr2 and the knockdown transformants to various cations in comparison to WT. A2 and A15 showed enhanced sensitivity to Aluminium (Al³⁺) (Table 1).

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Table 1. Altered Cation Sensitivity in the knockdown transformants.

https://doi.org/10.1371/journal.pone.0159244.t001

This is consistent with the observation in S. cerevisiae that over-expression of the Mg²⁺ transporter provides resistance to Al³⁺, justifying the ALR (Aluminium Resistance) nomenclature. The knockdown transformants were also more sensitive to Copper (Cu²⁺) and Iron (Fe³⁺) (Table 1). Their increased sensitivity towards these cations suggests that the level of Mg²⁺ required to provide resistance against these cations is dependent on MoALR2 function. Conversely, A2 and A15 were more resistant to Nickel (Ni²⁺), Cobalt (Co²⁺), Zinc (Zn²⁺) and Manganese (Mn²⁺) (Table 1). The Δmnr2 knockout, on the other hand, showed greater sensitivity to Ni²⁺, Co²⁺, Zn²⁺ and Mn²⁺. These cations have been reported to be transported by CorA transporters [26, 27], so the higher resistance of the knockdown transformants to them is likely to be due to reduced uptake by the lower number of Mg²⁺ transporters. This is also supported by the intracellular levels of Zn²⁺ in A2 and A15, in which Zn²⁺ levels were reduced to 78% and 61% of WT levels (S3A Fig).

CorA transporters are required for mycelial growth and surface hydrophobicity in M. oryzae

We next set out to evaluate the effect of reduced Mg²⁺ transport on development in M. oryzae. Δmnr2 showed ~5% reduction in colony diameter as compared to WT and failed to produce melanin in the aerial hyphae (S3B Fig). The MoALR2 knockdown transformants showed ~6% to 25% reduction in growth on Oat Meal Agar (OMA) medium (S3B Fig) (S3 Table) and this reduction was correlated with the degree of silencing of MoALR2. In comparison to WT, Δmnr2 formed fewer aerial hyphae, while the knockdown transformants showed very sparse aerial hyphae, as seen in A2 and A15 (Fig 6A).

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Fig 6. CorA transporters are required for mycelial growth and surface hydrophobicity in M. oryzae.

(A) Microscopic examination of the hyphal growth of WT, Δmnr2 and knockdown transformants was assessed. Pictures were taken after 2dpi grown on 0.8% agarose. (B) Ability of WT spores to form vegetative hyphal growth following germination was assessed on OMA (left) and YEGA (right) with different concentrations of EDTA. 10μl of spores with increasing dilution was spotted onto the plates. The ability of Mg²⁺ to restore the germination capability of spores was also checked on Mg²⁺ supplemented medium in presence of EDTA. (C) 10 μl of water or detergent solution containing 0.02% SDS+5mM EDTA were placed on the surfaces of the WT, Δmnr2 and knockdown transformants and photographed after 1 min. (D) mRNA levels of MoMPG1 and MoMPG2 were estimated by qRT-PCR in Δmnr2 and knockdown transformants. All transcript levels were normalized to that of WT. (E) mRNA levels of MoMPG1 and MoMPG2 were estimated by qRT-PCR at two different concentrations of extracellular Mg²⁺ in WT. All transcript levels were expressed as relative values, with 1 corresponding to levels at 4mM. Error bar denote SD. ** means P value at <0.0001 and * means significant at P value <0.05. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g006

To investigate whether similar growth defects are also observed in situations of low Mg²⁺ availability in M. oryzae, growth of WT was assayed in Mg²⁺ limiting conditions, using EDTA to lower Mg²⁺ availability. Growth on OMA was severely reduced with increasing concentrations of EDTA, being retarded significantly at 0.5mM EDTA (S4A Fig). On supplementing this medium with 50mM and 250mM extracellular Mg²⁺, growth was restored to normal (S4B Fig). At a concentration of 0.6mM EDTA, there was complete growth inhibition. Under 0.5mM EDTA stress, WT frequently formed sectored colonies with certain sectors showing phenotypic differences (less melanized whitish sectors and melanized grayish sectors). Given the frequency with which such sectors appeared, it is likely that their altered phenotypes were due to epigenetic changes. When the phenotypically different sectors (grown on OMA without EDTA) were grown again in the absence of 0.5mM EDTA stress, these differences (less melanized and whitish) persisted, as observed up to 6 sub-culturings (S4B Fig).

Vegetative hyphal growth from WT spores following germination was checked on OMA and YEGA with increasing concentrations of EDTA. At 0.3mM EDTA growth from spores was severely restricted, while 50mM Mg²⁺ could rescue the growth defect (Fig 6B).

Hydrophobins are surface proteins produced by filamentous fungi that are important for growth of aerial hyphae, hyphal surface hydrophobicity and attachment to solid supports [28–31]. Reduced surface hydrophobicity leads to a “wettable” phenotype where water droplets are not retained as beads on aerial hyphae. Such a wettable phenotype has been observed previously in M. oryzae in hydrophobin (MoMHP1, MoMPG1) and phosphodiesterase (MoPDEH) mutants [6, 32–34]. Δmnr2 and MoALR2 knockdown transformants were tested for their ability to retain drops of water and detergent solution to assess effects of MoALR2 and MoMNR2 on surface hydrophobicity. Compared to WT, Δmnr2 showed a wettable phenotype both with water and detergent solution, but could hold water longer than the MoALR2 knockdown transformants, which showed an easily wettable phenotype (did not retain solution at all) (Fig 6C). Thus, while MoMNR2 has some effect on surface hydrophobicity, MoALR2 appears to be the critical determinant. To investigate whether this wettable phenotype was mediated through hydrophobins, we measured the expression levels of hydrophobins, MoMPG1 (Mgg_10315) and MoMPG2 (Mgg_01173), in the Δmnr2 and knockdown transformants. There was substantial decrease in the expression of MoMPG1 in the knockdown transformants and in A15 the levels decreased by ~95%. The levels of MoMPG2 did not change significantly (at P<0.0001) (Fig 6D).

To check whether extracellular Mg²⁺ availability affects expression of MoMPG1 and MoMPG2, their transcript levels in WT were studied at different concentrations of Mg²⁺ and in presence of EDTA. In presence of EDTA, the expression of MoMPG1 decreased to as little as 10% while MoMPG2 still showed 60% expression (significant at P<0.0001) (Fig 6E). At 50mM and 250mM Mg²⁺ the expression of MoMPG1 and MoMPG2 was similar to that of control at 4mM. Thus, we show for the first time that in M. oryzae, decrease in Mg²⁺ levels, either by silencing of transporter function (in the knockdown transformants), or by using EDTA (in WT), has a direct effect on the expression of both hydrophobins, especially MoMPG1.

Hyphal growth on OMA was observed at regular time intervals post inoculation. As early as 12 days post inoculation (dpi), Δmnr2 and MoALR2 knockdown transformants displayed autolysis at the centre of the colony (Fig 7A). WT did not show any such phenotype even up to three weeks. The autolysis was more severe in transformants with MoALR2 expression below 50%, namely, WT+siALR2_48, A2 and A15 (Fig 7A). We followed Δmnr2 for a longer time period under different extracellular Mg²⁺ concentrations. Though at 12dpi Δmnr2 showed autolysis only at the centre, by 16 dpi, autolysis had spread to include a large proportion of the Δmnr2 colony. 50mM and 250mM extracellular Mg²⁺ supplement delayed the onset of autolysis and as a result the autolysis area observed at 16 dpi was reduced (Fig 7B). On the contrary, EDTA hastened the process with Δmnr2 displaying the phenotype even at 11 dpi with increasing severity on subsequent days (Fig 7B). In spite of wild type levels of MoALR2, Δmnr2 showed early autolysis compared to WT, suggesting that MoMNR2 is essential for long term survival of M. oryzae.

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Fig 7. CorA transporters are required for preventing autolysis.

(A) WT, Δmnr2 and knockdown transformants were grown on OMA for 12 days. Early autolysis was monitored compared to WT. (B) WT and Δmnr2 were grown on OMA supplemented with 4mM, 50mM, 250mM extracellular Mg²⁺ and 0.3mM EDTA. Autolysis was monitored from 10 dpi to 16 dpi and area under autolysis was measured at 16dpi.

https://doi.org/10.1371/journal.pone.0159244.g007

Magnesium uptake by CorA transporters is essential for progression of the infection cycle in M. oryzae

The ability of pathogenic fungi to sporulate is critical to the spread of infection. Δmnr2 knockout showed a 23% reduction in spore count. In the MoALR2 knockdowns, sporulation efficiency decreased with a reduction in the expression of MoALR2, to as low as 20% in WT+siALR2_48 (Fig 8A). A2 and A15 completely failed to sporulate, suggesting that maintenance of MoALR2 levels is critical for conidiogenesis.

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Fig 8. CorA transporters are required for Sporulation and Appressorium formation.

(A) Ability of Δmnr2 and knockdown transformants to sporulate was checked on OMA 8 days post inoculation and quantified. Aerial hyphal and conidial developmentwere also assessed for Δmnr2 and knockdown transformants at 48 hpi. (B), (C) Appressorial assay for Δmnr2 and knockdown transformants was performed on hydrophobic gelbond film and the ability to form infection structure was assessed and quantified at 6 hours and 12 hours (CO-Conidium, GT-Germ tube, AP-Appressorium). The values are represented as percentage of spore (ungerminated), germ tube and appressoria formed at the given time interval. Mycelial blocks were placed on hydrophobic surface and incubated upto 72 hours at 28°C for non-sporulating transformants. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g008

We studied the ability of WT to sporulate in presence of EDTA. Sporulation increased up to 0.3mM EDTA as compared to control (OMA with no EDTA), and then decreased at higher concentrations (S5A Fig). At 0.5mM EDTA, sporulation was severely decreased. Mg²⁺ supplementation of 250mM was able to rescue this decrease in the sporulation at 0.5mM EDTA, suggesting that adequate Mg²⁺ levels are required for sporulation.

During infection, the spores germinate and differentiate into appressoria. To evaluate the role of MoALR2 and MoMNR2 in germination and appressorium formation in WT and knockdown transformants, percentage of spores that germinated and formed appressoria at 6 and 12 hours was determined (Fig 8B and 8C). While in WT 90% of spores had germinated by 12 hours, in knockdown transformants the percentage of ungerminated spores ranged from 33% in Δmnr2+siALR2_79 to 41% in WT+siALR2_48. In WT, the percentage of appressoria formed increased from 41% at 6 hours to 85% at 12 hours. In Δmnr2 the percentage of appressoria formed at 12 hours was 83%, which is comparable to WT. In the MoALR2 knockdown transformant WT+siALR2_48, the percentage of appressoria formed at 12 hours was only 33%. The percentage of spores that failed to germinate and form appressoria increased with higher level of silencing in the knockdown transformants (Fig 8C). Since A2 and A15 failed to sporulate, mycelial plugs from these transformants were inoculated on hydrophobic surface and incubated under moist conditions for 48 hours (as mycelial tips are also capable of forming appressoria-like structures). The mycelial tips of A2 and A15 failed to develop any appressorium-like structure of the kind seen in the WT (Fig 8B). Thus it is evident that a minimum level of MoALR2 expression is critical for appressorium formation from germinated spores as well as from hyphae.

To test the ability of the conidia to germinate and form appressoria in presence of EDTA and EDTA with 50mM Mg²⁺ supplementation, appressorium formation was studied at different time points in WT (Fig 9A). In the presence of 0.25mM EDTA, even at 24 hours the spores failed to form appressoria (for every time point n>100). Most of the spores (63%) were stalled in the germ tube stage (S5B Fig). Extracellular Mg²⁺ (50mM) was able to rescue the defect in appressorium formation in presence of EDTA in WT. At 24 hours, approximately 53% of the spores had formed appressoria in presence of 50mM Mg²⁺ and 0.25mM EDTA (S5B Fig).

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Fig 9. Mg²⁺ uptake by CorA transporters is essential for progression of the infection cycle.

(A) Ability to form appressoria in water, 0.25mM EDTA and 0.25mM EDTA+50mM Mg²⁺ was observed at different time intervals in WT tagged with H1:RFP and Tub:GFP. (B) Detached rice leaves of cultivar HR12 were inoculated with spores (1x10⁴/ml) and mycelial plugs. Disease symptoms (lesions) were assessed 4 days post inoculation. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g009

Rice detached leaf infection test showed that the severity of infection decreased in the knockdown transformants (Fig 9B), where small brown lesions were observed as compared to the typical brown bordered gray centered lesions of the WT. The decrease was consistent with the decrease in the levels of MoALR2, indicating the importance of MoALR2 for pathogenicity in M. oryzae. Thus, extracellular Mg²⁺ availability and transport are critical at all stages of the infection cycle including hyphal growth, conidiation, spore germination, appressorium formation and disease progression in M. oryzae.

MoALR2 affects intracellular cAMP levels in M. oryzae

In M. oryzae, cAMP mediated signaling through MoPMK1 (Mgg_09565) is crucial for conidiation and appressorium initiation [7, 35]. cAMP is synthesized by Mg²⁺ dependent adenylate cyclase [14, 36]. In view of the low Mg²⁺ levels in the knockdown transformants, we looked for changes in intracellular cAMP levels. The levels of cAMP in Δmnr2 and the knockdown transformants ranged from 97to 20 fmol mg^-1 as compared to 105 fmol mg^-1 in WT (Fig 10A). The cAMP levels correlated with the transcript levels of MoALR2 and intracellular Mg²⁺ levels in the transformants. In WT, cAMP levels decreased in presence of EDTA (Fig 10B) and were consistent with the intracellular levels of Mg²⁺ in presence of EDTA described earlier (Fig 2D). cAMP levels were restored at 50mM Mg²⁺ at 8 hours in WT (Fig 10B). In presence of EDTA, cAMP levels in A2 and A15 did not correlate to intracellular Mg²⁺ levels (data not shown).

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Fig 10. MoALR2 affects intracellular cAMP levels and cAMP mediated signaling in M. oryzae.

(A) Intracellular cAMP levels were estimated in WT, Δmnr2 and knockdown transformants. The bar graph represents cAMP levels in fmol mg^-1. (B) Estimation of intracellular levels of cAMP at 50mM extracellular Mg² in the double knockdown transformants, A2 and A15 in presence of 4mM Mg²⁺, 4mM Mg²⁺ + 4mM EDTA and at 50mM Mg²⁺. The values are expressed in fmol mg^-1. (C), (D) mRNA levels of MoMAC1 and MoPMK1 were estimated by qRT-PCR in WT, Δmnr2 and knockdown transformants. (E), (F) mRNA levels of MoMAC1 and MoPMK1 were estimated by qRT-PCR at two different concentrations of extracellular Mg²⁺ in WT, A2 and A15. The transcript levels were normalized to that of WT. Error bar denote SD. ** means P value at <0.0001 and * means significant at P value <0.05. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g010

We studied the expression of the adenylate cyclase gene, MoMAC1 (Mgg_09898) and of MoPMK1 (encoding MAPK) in the knockdown transformants and found significant decrease in expression, with the transcript levels decreasing to 43% and 20% respectively in A15 (Fig 10C and 10D). To study whether extracellular Mg²⁺ affects expression of MoMAC1 and MoPMK1, we looked at their transcript levels in A2 and A15 with 50mM Mg²⁺ supplementation, and found that expression was restored to WT levels (Fig 10E and 10F). Since the knockdown transformants of MoALR2 showed decreased ability to conidiate and form appressoria, the low level of intracellular cAMP is likely to be one of the factors contributing to the defect seen in appressorium formation in knockdown transformants.

MoALR2 knockdown transformants show altered cell wall integrity

Mg²⁺ is vital for the integrity of the cell wall and cell membrane [37, 9–12]. We asked whether decreased Mg²⁺ levels lead to changes in cell wall integrity (CWI). Growth was measured on media containing the cell wall stressors Congo Red (CR) [38] and Caffeine [39–41]. Δmnr2 transformants were resistant to 1.5 and 2 mg ml^-1 CR, similar to WT. However the knockdown transformants, both in WT and Δmnr2 backgrounds, were more sensitive to CR, with A15 showing the maximum sensitivity (Table 2).

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Table 2. Cell Wall Integrity assay.

https://doi.org/10.1371/journal.pone.0159244.t002

The degree of sensitivity towards cell wall stressors increased in proportion to silencing of MoALR2. Mg²⁺ supplementation could restore the normal (lower) sensitivity to cell wall stressors (S6 Fig). These results indicate that maintenance of Mg²⁺ levels dependent on MoALR2 is critical for the integrity of the cell wall.

To understand the cell wall defects in the knockdown transformants better, we looked at expression of cell wall maintenance related genes. We found decreased expression of two chitin synthase genes, MoCHS1 (Mgg_01802) and MoCHS4 (Mgg_09962), in the knockdown transformants (Fig 11E).

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Fig 11. Expression analysis of genes involved in the CWI Pathway in knockout and knockdown transformants.

(A) mRNA levels of MoGTBP1, MoPKC1, MoMAPK3, MoMKK1 and MoMPS1 were estimated by qRT-PCR. (B) mRNA levels of MoCRZ1 and MoFKS1 were estimated by qRT-PCR. (C) mRNA levels of MoMPS1 were estimated by qRT-PCR at two different concentrations of extracellular Mg²⁺ in WT, A2 and A15. (D) mRNA levels of MoABP1 and MoMTI1 were estimated by qRT-PCR. (E) mRNA levels of MoCHS1 and MoCHS4 were estimated by qRT-PCR. All transcript levels were normalized to that of WT. Error bar denote SD. ** means P value at <0.0001 and * means significant at P value <0.05. The experiments were repeated in triplicate, N = 3.

https://doi.org/10.1371/journal.pone.0159244.g011

We studied the expression of genes involved in the CWI pathway in Δmnr2 and knockdown transformants. In simultaneously silenced transformants (for MoALR2 and MoMNR2), A2 and A15, there was significant down-regulation of genes involved in the Pkc1 activated mitogen activated protein (MAP) kinase cascade including MoPKC1, MoMKK1,MoMAPK3 and MoMPS1 (at P<0.05) (Fig 11A) only forMoGTBP (Mgg_07176) (Rho1), did expression not change significantly (at P<0.0001). In contrast Δmnr2 showed increased expression of MoPKC1 and MoMKK1. Increased expression of MoMKK1 was also observed in Δmnr2+siALR2_79 and Δmnr2+siALR2_66 (MoALR2 silencing in Δmnr2 background), while no significant changes were observed in WT+siALR2_56 and WT+siALR2_48 (MoALR2 silencing in WT background).

The expression of downstream effectors of CWI signaling, MoFKS1 (Mgg_00865) and MoCRZ1 (Mgg_05133) was also significantly decreased in the knockdown transformants (Fig 11B). The expression of MoMPS1 in A2 and A15 was restored to WT levels when supplemented with 50mM Mg²⁺ (Fig 11C), indicating that Mg²⁺ levels affect the expression of MoMPS1. The decreased expression of CWI signaling genes in the knockdown transformants explains the sensitivity towards cell wall stress and demonstrates the role of MoALR2 in maintenance of Mg²⁺ levels critical for cell wall integrity.

Actin cytoskeletal reorganisation is an important aspect of the compensatory response to cell wall defects [37]. We analyzed the expression of the genes MoABP1 (Mgg_06358) and MoMTI1 (Mgg_04116), in Δmnr2 and the knockdown transformants by qRT-PCR. There was significantly increased expression of both MoABP1 and MoMTI1 in A2 and A15 (at P<0.05) (Fig 11D). The expression of MoABP1 was 15 fold higher, implying that MoAbp1 might play a role in stabilizing the cytoskeleton and membranes to compensate for the lack of Mg²⁺, which is known to be crucial for maintenance of cell shape and membrane integrity.

Fungal strain and culture conditions

Magnaporthe oryzae B157 strain (MTCC accession number 12236), belonging to the international race IC9 was previously isolated in our laboratory from infected rice leaves [51]. The Δku80 mutant used in the present study was generated by replacing MoKU80 ORF with Zeocin selection marker in wild type B157 strain (WT) in our laboratory. The fungus was grown and maintained on YEG medium (Glucose1 g, yeast extract 0.2 g, H₂O to 100 ml) or Oatmeal agar (Hi-Media, Mumbai, India).

Complementation of a S. cerevisiae Δalr1Δalr2 mutant

S. cerevisiae Δalr1Δalr2 mutant (CM66) having the genotype Mata alr1::HIS3, alr2::TRP1, his3-200, ura3-52, leu2-1, lys2-202, trp1-63 and the strain from which it was derived (CM52) having the genotype Mata his3-200, ura3-52, leu2-1, lys2-202, trp1-63 was used for functional complementation studies. The full length gene of MoALR2 (1.9kb) was amplified from genomic DNA and cloned at PvuII site in the yeast episomal vector pYES2 (Invitrogen, California, USA) to generate pYES2-MoALR2. The full length gene of MoMNR2 (2.5kb) was amplified from genomic DNA and cloned first at EcoRV site in pBluescript KS (+). The full length gene was moved into pYES2 vector at HindIII and BamHI site under Gal1 promoter to give pYES2-MoMNR2. For MoMNR2_489-812 cloning, the CorA domain of MoMNR2 was PCR amplified from genomic DNA and cloned first in pBluescript KS (+) at EcoRV site. The CorA domain of MoMNR2 was moved into pYES2 vector at XhoI and BamHI site under Gal1 promoter to give pYES2-MoMNR2_489-812. This construct was used to transform the S. cerevisiae Δalr1Δalr2 double mutant (CM66). Colonies were selected on SD medium lacking uracil and having lysine, leucine, 2% Galactose and 500mM MgSO₄. Transformed colonies obtained were grown in SD media (lysine + leucine + 2% Galactose + 500mM MgSO₄) till saturation and cells were spotted on SD media having leucine, lysine, 2% Galactose and 4mM MgSO₄/500mM MgSO₄. The growth of colonies was seen 4 days post inoculation at 28°C. All primers used in the study are listed in S4 Table.

Raising of antibodies againstMoAlr2 and MoMnr2

The CorA domains of MoAlr2 and MoMnr2 have 50% identity at the protein level and the size of the proteins is 70kDa and 90kDa respectively. The CorA domain from MoMNR2 was amplified (1kb) from genomic DNA as it has no intron. The amplified product was cloned in pBluescript KS (+) vector at EcoRV site and then was ligated in bacterial expression vector pET30a (+) vector at NdeI and KpnI site translationally in frame with a (His)₆ tag at the C terminus. E. coli BL21 DE3 cells were transformed with the protein expression construct. The transformed colonies were grown in Luria Bertani (LB) medium O/N. 1% inoculum was used the next day and the culture was grown to an O.D. (λ₆₀₀) of 0.4 to 0.6 O.D.1mM IPTG was used for induction of the protein for another 4 hours. The induced protein having Polyhistidine at the carboxyl terminus was purified using Ni-NTA affinity chromatography (Novagen, Darmstadt, Germany) according to manufacturer’s protocol. The purified protein was used to raise polyclonal antibodies in New Zealand White Rabbit. Antibody titer was estimated by indirect-enzyme linked immunosorbent analysis (ELISA) using HRP conjugated anti-rabbit IgG (Bangalore Genie, Bangalore, India) as the secondary antibody.

Indirect immunolocalization of MoAlr2 and MoMnr2 in M. oryzae

The wild type B157 spores were fixed with 10% formaldehyde, 5% acetic acid, and 85% ethanol for 30 minutes at room temperature and the fixed sample was incubated in PBS + 0.1% Triton X-100 for 2–5 minutes. The sample was given wash with PBS for 10 to 15 minutes. The fixed samples were further treated as described [52] with few modifications. Primary antibodies used were against CorA domain of MoMnr2. Secondary antibody, TRITC-conjugated anti-rabbit IgGs raised in goat, (Sigma Chemical Co, St Louis, MO, USA) diluted to 1:20 in PBS was used for staining for 2 hours. Three washes each of 15 minutes were given with PBST which was followed by vacuolar staining with Oregon green 488 carboxylic acid diacetate (cDFFDA) (Molecular Probes, Invitrogen, California, USA) for 10 minutes followed by three washes with PBST. The slides were observed under 63X using LSM 700 microscope (Carl Zeiss, Jena, Germany) at 557nm excitation and 576nm emission for TRITC and at 501nm excitation and 526nm emission for Oregon green 488. Image analysis was done using ZEN software.

Plasmid construction and transformation of M. oryzae

The vector pGKO2-MoALR2 was constructed for carrying out targeted disruption in WT. Full length gene of MoALR2 (1.9kb) was PCR amplified, end filled and cloned at EcoRV site in pBluescript KS (+) to give KS-MoALR2. The HPT cassette used for disrupting the gene MoALR2 was taken out from pAN7.1 [27] vector using BglII and HindIII having glyceraldehyde 3 phosphate dehydrogenase (gpdA) promoter, end filled and cloned at EcoRV site (present in between the MoALR2 gene). The whole disruption cassette (~6kb) was moved from pBluescript KS (+) and cloned into a binary vector pGKO2 at KpnI and SpeI site. The A. tumefaciens strain LBA4404/pSB1 was first transformed with pGKO2-MoALR2-HPT via triparental mating (Helper plasmid pRK2013). The transformed Agrobacterium was then used to carry out A. tumefaciens mediated transformation of M. oryzae as described [53]. The transformants were selected on Hygromycin (200μg ml^-1) and 5-fluoro-2’-deoxyuridine (5μM). We attempted several rounds of transformation to disrupt MoALR2 using ATMT. Protoplast transformation was also carried out with the full disruption cassette and by split marker technique (two different overlap regions). Further, supplementation of different Mg² concentrations during selection was done to overcome the selective disadvantage facing slow growing mutants. A smaller disruption cassette of MoALR2(~4kb) was also made in the vector pBluescript KS (+) where KS-MoALR2 was digested with EcoRV (site present in between the gene) and HPT cassette of 2kb was PCR amplified from pSilent and cloned at EcoRV site in KS-MoALR2 to give pBSKS-MoALR2-HPT. This cassette was used to carry out transformation of the Δku80 strain. Targeted deletion of MoMNR2 was carried out by transforming WT protoplast with a knockout construct which was obtained by double-joint PCR [54], where ~1kb flanking sequence of MoMNR2 was amplified and fused to Zeocin resistance cassette. Transformants were selected on Zeocin (300μg ml^-1). They were screened with PCR using different sets of primer combinations and confirmed for target gene replacement by Southern blot analysis using both the flanking sequences as probes.

For knockdown of MoALR2 a ~110 bp fragment of MoALR2, which spans the siRNA matching to the 5’ UTR, was amplified and cloned in pSilent-Dual 2 vector [5] at SmaI site. For simultaneous silencing of MoALR2 and MoMNR2, the full length gene of MoALR2 cloned in pBluescript KS (+) was digested with KpnI and BamHI to give a fragment of 1.4kb (having a portion of the CorA domain) and was cloned in anti-sense orientation in pSilent-1 [55] at KpnI and BglII site. For RNAi approach, the full length gene of MoALR2 cloned in pBluescript KS (+) was digested with HindIII and 525bp fragment (spanning CorA domain) was cloned in pSilent-Dual 2 vector at HindIII site. The knockdown constructs were used for protoplast transformation of WT as described [31]. Putative knockdown transformants were selected on Hygromycin (200μg ml^-1) and Geneticin (1mg ml^-1). Untransformed WT was kept as a control which did not grow on either Hygromycin or Geneticin medium. Vector transformation (pSD2) was also done as a control. The transformants were maintained as monoconidial isolates to obtain pure cultures. The knockdown transformants were screened by PCR and confirmed by Southern hybridization.

Nucleic acid manipulation and Southern Blotting

Fungal genomic DNA was extracted as described by Dellaporta et al. [56]. Southern blot analysis was carried out as previously described Sambrook et al. [57]. In case of Δmnr2 and WT, genomic DNA was digested with HindIII, EcoRI and EcoRV and the blot was probed first with a 1kb upstream fragment of MoMNR2 and then re-probed with a 1kb downstream fragment of MoMNR2. The knockdown transformants for MoALR2 were digested with EcoRI and probed with 1.2kb upstream fragment of MoALR2. The simultaneously silenced transformants, A2 and A15 was digested with SalI and the blot was probed with TrpC promoter (~400bp). The probes were labeled and hybridizing bands were detected using Gene Images AlkPhos Direct Labeling and Detection system as per manufacturer’s instructions (Amersham, Buckinghamshire, England).

Quantitative Real Time PCR analysis for gene expression

Fungus was grown in Complete Medium (CM) for 72 hours (when no treatment was given to the biomass), else it was grown in CM for 48 hours, followed by two washes of the biomass with sterile milliQ water. The fungus was then transferred into Minimal Medium for 24 hours after which it was given treatments of Mg²⁺ or Ca²⁺ for the given time period. Fungal biomass was harvested and frozen in liquid nitrogen. Total RNA was isolated using TRIzol reagent (Invitrogen Life Technologies, California, USA). 2μg of total RNA was reverse transcribed into first strand cDNA using oligo (dT) primer and M-MuLV Reverse transcriptase (NEB, Massachusetts, USA). Quantitative PCR was performed by monitoring in real time the increase in fluorescence of the SYBR Green dye either on a Light Cycler system for real-time PCR (Roche Applied Science, Mannheim, Germany)or ABI 7900 HT real time PCR (Applied Biosystems, California, USA), according to the manufacturer’s instructions. Thermal cycling conditions consisted of 10 minutes at 95°C 1 cycle, followed by 40 cycles of 10 seconds at 95°C, 10 seconds at 55°C and 15 seconds at 72°C for SYBR chemistry. Also Taqman Probes (Applied Biosystems, California, USA) specific for gene(s) were used to validate and study expression profile quantitatively. Reaction conditions were according to manufacturer’s instructions. Thermal cycling conditions consisted of 2 minutes at 50°C 1 cycle, 10 minutes at 95°C 1 cycle, 15 seconds at 95°C and 1 minute at 60°C 40 cycles. Each qRT-PCR quantification was carried out in triplicate and every biological repeat was kept in duplicate. To compare the relative abundance of target gene transcripts, the average threshold cycle (Ct) was normalized to that of GPDH gene for each of the treated samples as2^-ΔCt, where ΔCt = (Ct_gene of interest—Ct_GPDH) and fold changes were calculated by 2^-ΔΔCt, where ΔΔCt = (Ct _{gene of interest}−Ct _GPDH) _{test condition}−(Ct_{gene of interest}−Ct _GPDH) _control. The transcript levels were expressed as relative values, with 1 corresponding to the Wild type (WT).

X-Ray Fluorescence (XRF) for element analysis

For determining the levels of elements (mainly Mg²⁺ and Ca²⁺) in the knockout and knockdown transformants X- ray Fluorescence Analysis (XRF) [58] was performed. WT, knockout and knockdown transformants were grown in CM for 48 hours. The fungal biomass was washed twice with sterile milliQ water and the biomass was transferred into Minimal media for 24 hours. Then the biomass was grown under different concentrations of Mg²⁺ (4mM, 50mM and 250mM) for another 6 hours. The fungal biomass was harvested, frozen in liquid nitrogen and ground to fine powder. The biomass was dried completely at 37°C for 3–4 days, after which the element analysis was done using Energy dispersive X-ray Fluorescence Spectrometer EDX-720/800HS (Shimadzu, Singapore).

Phenotypic Characterization of transformants

Vegetative growth of the knockdown and knockout transformants was measured on OMA and YEGA 5 days post inoculation. The experiments were performed with replicates in three independent experiments.

The ability to produce conidia was measured by counting the numbers of conidia for the knockdown and knockout transformants isolated from the surface. Quantification of conidia was done using a hemacytometer (Marienfeld Superior, Lauda-Konigshofen, Germany).

For appressorium formation, equal number of spores were inoculated on hydrophobic surface, Gelbond film (Amersham Pharmacia Biotech AB, Uppsala, Sweden) and incubated under moist conditions for 6 and 12 hours. For non-sporulating knockdown transformants, mycelial plugs from actively growing transformants (5^th day plate) were inoculated and incubated under moist conditions for 48 hours. Appressorium formation in the knockdown and knockout transformants was checked at 40X magnification (Olympus, Tokyo, Japan). The relative percentage of appressoria formed was calculated for each time interval.

For assaying the sensitivity of the knockdown and knockout transformants towards CorA specific inhibitor (Cobalt(III) hexaammine(Co(III)Hex), a concentration ranging from 300μM to 400μM of Cobalt(III) hexaammine was added to YEG medium. The sensitivity was assessed 5 days post inoculation and radial growth was measured for these transformants.

Surface hydrophobicity assay

The knockout and knockdown transformants were tested for defects in surface hydrophobicity with water and detergent solution (0.02% SDS+5mM EDTA). 5 day old fungal culture grown on YEGA was inoculated with water and detergent solutions. The wettability of the transformants was checked by the extent to which water or detergent was retained on mycelia compared to WT.

Western blotting

Total protein was extracted from WT and knockdown transformants grown in CM for 72 h (when no treatment was given to the biomass); else the fungus was grown in CM for 48 hours, which was followed by two washes of the biomass with sterile milliQ water. The fungus was then transferred into Minimal medium for 24 hours after which the fungus was given treatments of Mg²⁺ or Ca²⁺ in the present study for the given time period. Fungal biomass was harvested and frozen in liquid nitrogen. Protein was extracted in Urea Buffer (9.5M urea, 2% v/v NP40, 5% βME) for 1 hour at RT. The concentration of the protein was estimated by Bradford method. The protein samples were electrophoresed on 10% SDS–polyacrylamide gel, followed by electro transfer to PVDF membrane (Hybond ECL, Amersham, Buckinghamshire, England). The immunoblots were developed with 1° antibody against MoMnr2 CorA domain and 2° antibody conjugated with HRP using 3’-3’- diamino Benzidin tetrahydrochloride dehydrate (Fluka, Washington DC, USA) detection method (Bangalore Genei, Bangalore, India) and Super Signal West Pico Chemiluminescent substrate (Thermo Scientific, Rockford, USA) as per the manufacturer’s instructions.

Cation sensitivity assay

For cation sensitivity assays, the knockout, knockdown transformants and WT were inoculated on YEGA having Zinc (Zn²⁺ 500μM), Cobalt (Co²⁺ 150μM), Manganese (Mn²⁺ 3mM), Iron (Fe³⁺ 2mM), Copper (Cu²⁺ 750μM), Aluminium (Al³⁺ 800μM), Nickel (Ni²⁺ 200μM). The sensitivity to different cations was studied by comparing the growth of transformants 5 days post inoculation with respect to WT. For all the sensitivity assays the minimum inhibitory concentration (MIC) was determined with respect to WT.

Infection assay and cell wall integrity assay

Leaves of 21 day old rice seedlings of HR-12 cultivar were used for inoculating spores or mycelial plugs (for non-sporulating transformants) of knockout and knockdown transformants and were placed on water agar with kinetin (2 mg l^-1). Disease symptoms were recorded after 3–4 days.

For the cell wall integrity assay, WT, knockout and knockdown transformants were inoculated on YEGA containing Congo Red (1.5mg ml^-1and 2mg ml^-1) or Caffeine (2.5mM and 3mM). Sensitivity to these cell wall stress molecules was studied by comparison of growth to that of WT 5 days post inoculation.

Quantification of intracellular cAMP levels

WT, knockout and knockdown transformants were grown in CM for 48 hours. The fungal biomass was washed twice with sterile milliQ water and then the biomass was transferred into Minimal media containing 4mM Mg²⁺ for 24 hours. The biomass was harvested and frozen. The fungal biomass was ground to fine powder in liquid nitrogen and after the liquid nitrogen was evaporated, equal weight of frozen biomass (0.1gm) was taken and homogenized in 300μl of 0.1N HCl. The sample was vortexed for 30 minutes, followed by centrifugation at 5000 x g for 15 minutes at room temperature. The supernatant was collected and 100μl of sample in each case was taken. Quantification of intracellular cAMP levels was carried out by cAMP Direct Immunoassay Kit as per manufacturer’s instructions (Calbiochem, Darmstadt, Germany).