Fungal Histidine Phosphotransferase Plays a Crucial Role in Photomorphogenesis and Pathogenesis in Magnaporthe oryzae

Two-component signal transduction (TCST) pathways play crucial roles in many cellular functions such as stress responses, biofilm formation, and sporulation. The histidine phosphotransferase (HPt), which is an intermediate phosphotransfer protein in a two-component system, transfers a phosphate group to a phosphorylatable aspartate residue in the target protein(s), and up-regulates stress-activated MAP kinase cascades. Most fungal genomes carry a single copy of the gene coding for HPt, which are potential antifungal targets. However, unlike the histidine kinases (HK) or the downstream response regulators (RR) in two-component system, the HPts have not been well-studied in phytopathogenic fungi. In this study, we investigated the role of HPt in the model rice-blast fungal pathogen Magnaporthe oryzae. We found that in M. oryzae an additional isoform of the HPT gene YPD1 was expressed specifically in response to light. Further, the expression of light-regulated genes such as those encoding envoy and blue-light-harvesting protein, and PAS domain containing HKs was significantly reduced upon down-regulation of YPD1 in M. oryzae. Importantly, down-regulation of YPD1 led to a significant decrease in the ability to penetrate the host cuticle and in light-dependent conidiation in M. oryzae. Thus, our results indicate that Ypd1 plays an important role in asexual development and host invasion, and suggest that YPD1 isoforms likely have distinct roles to play in the rice-blast pathogen M. oryzae

Dual role for fungal-specific outer kinetochore proteins during cell cycle and development in Magnaporthe oryzae

The outer kinetochore DASH complex (also known as the Dam1 complex) ensures proper spindle structure and chromosome segregation. While DASH complex protein requirement diverges among different yeasts, its role in filamentous fungi has not yet been investigated. We studied the dynamics and role of middle (Mis12) and outer (Dam1 and Ask1) kinetochore proteins in the filamentous fungal pathogen, Magnaporthe oryzae, which undergoes multiple cell cyclelinked developmental transitions. While Mis12 was constitutively present in the nucleus, Dam1 and Ask1 were recruited only during mitosis. Although Dam1 was not required for viability, loss of its function (dam1 Delta) delayed mitotic progression, resulting in impaired conidial and hyphal development. Both Dam1 and Ask1 also localised to the hyphal tips, in the form of punctae oscillating back and forth from the growing ends, suggesting that Magnaporthe DASH complex proteins may play a non-canonical role in polarised growth during interphase, in addition to their function in nuclear segregation during mitosis. Impaired appressorial (infection structure) development and host penetration in the dam1. mutant suggest that fungus-specific Dam1 complex proteins could be an attractive target for a novel anti-fungal strategy

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

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