XRF analysis of knockdown transformants.

<p>(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.</p

CorA transporters are required for preventing autolysis.

<p>(A) WT, <i>Δmnr2</i> and knockdown transformants were grown on OMA for 12 days. Early autolysis was monitored compared to WT. (B) WT and <i>Δmnr2</i> 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.</p

CorA transporters are required for Sporulation and Appressorium formation.

<p>(A) Ability of <i>Δmnr2</i> and knockdown transformants to sporulate was checked on OMA 8 days post inoculation and quantified. Aerial hyphal and conidial developmentwere also assessed for <i>Δmnr2</i> and knockdown transformants at 48 hpi. (B), (C) Appressorial assay for <i>Δmnr2</i> 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.</p

CorA Mg²⁺ transporters affect metal ion composition in <i>M</i>. <i>oryzae</i>.

<p>(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, <i>Δmnr2</i> and knockdown transformants. (B) mRNA levels of <i>MoALR2</i> and <i>MoMNR2</i> 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.</p

Regulation of <i>MoALR2</i> in WT at mRNA and protein level with respect to extracellular Ca²⁺.

<p>(A) mRNA levels of <i>MoALR2</i> 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.</p

Altered Cation Sensitivity in the knockdown transformants.

<p>Altered Cation Sensitivity in the knockdown transformants.</p

Regulation of <i>MoALR2</i> and <i>MoMNR2</i>at mRNA and protein level with respect to extracellular Mg²⁺.

<p>(A) mRNA levels of <i>MoALR2</i> were estimated in WT by qRT-PCR at different concentrations of extracellular Mg²⁺. (B) mRNA levels of <i>MoMNR2</i> 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.</p

Magnesium Uptake by CorA Transporters Is Essential for Growth, Development and Infection in the Rice Blast Fungus <i>Magnaporthe oryzae</i>

<div><p><i>Magnaporthe oryzae</i>, the causative organism of rice blast, infects cereal crops and grasses at various stages of plant development. A comprehensive understanding of its metabolism and the implications on pathogenesis is necessary for countering this devastating crop disease. We present the role of the CorA magnesium transporters, MoAlr2 and MoMnr2, in development and pathogenicity of <i>M</i>. <i>oryzae</i>. The <i>MoALR2</i> and <i>MoMNR2</i> genes individually complement the Mg²⁺ uptake defects of a <i>S</i>. <i>cerevisiae</i> CorA transporter double mutant. <i>MoALR2</i> and <i>MoMNR2</i> respond to extracellular Mg²⁺ and Ca²⁺ levels and their expression is elevated under Mg²⁺ scarce conditions. RNA silencing mediated knockdown of <i>MoALR2</i> (WT+siALR2, <i>Δmnr2</i>+siALR2 and <i>ALR2</i>+<i>MNR2</i> simultaneous silencing) drastically alters intracellular cation concentrations and sensitivity to metal ions. <i>MoALR2</i> silencing is detrimental to vegetative growth and surface hydrophobicity of mycelia, and the transformants display loss of cell wall integrity. <i>MoALR2</i> is required for conidiogenesis and appressorium development, and is essential for infection. Investigation of knockdown transformants reveal low cAMP levels and altered expression of genes encoding proteins involved in MoMps1 cell wall integrity and cAMP MoPmk1 driven MAP Kinase signaling pathways. In contrast to <i>MoALR2</i> knockdowns, the <i>MoMNR2</i> deletion (<i>Δmnr2</i>) shows increased sensitivity to CorA inhibitors as well as altered cation sensitivity, but has limited effect on surface hydrophobicity and severity of plant infection. Interestingly, <i>MoALR2</i> expression is elevated in <i>Δmnr2</i>. Impairment of development and infectivity of knockdown transformants and altered intracellular cation composition suggest that CorA transporters are essential for Mg²⁺ homeostasis within the cell, and are crucial to maintaining normal gene expression associated with cell structure, signal transduction and surface hydrophobicity in <i>M</i>. <i>oryzae</i>. We suggest that CorA transporters, and especially <i>MoALR2</i>, constitute an attractive target for the development of antifungal agents against this pathogen.</p></div

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

<p>(A) mRNA levels of <i>MoGTBP1</i>, <i>MoPKC1</i>, <i>MoMAPK3</i>, <i>MoMKK1</i> and <i>MoMPS1</i> were estimated by qRT-PCR. (B) mRNA levels of <i>MoCRZ1</i> and <i>MoFKS1</i> were estimated by qRT-PCR. (C) mRNA levels of <i>MoMPS1</i> were estimated by qRT-PCR at two different concentrations of extracellular Mg²⁺ in WT, A2 and A15. (D) mRNA levels of <i>MoABP1</i> and <i>MoMTI1</i> were estimated by qRT-PCR. (E) mRNA levels of <i>MoCHS1</i> and <i>MoCHS4</i> 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.</p