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

Abstract

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.

Keywords: His-Asp phosphorelay; Magnaporthe oryzae; conidiation; host invasion; light response; rice blast; two-component system.

Figure 1

Identification of Ypd1 and its gene isoforms in M. oryzae. (A) A dendrogram showing phylogenetic analysis of the Ypd1 sequences from various filamentous and non-filamentous fungi. Arrowhead denotes Ypd1 from M. oryzae. (B) Multiple sequence alignment of Ypd1 sequences from different fungi. The red box marks the highly conserved catalytic region in the Ypd1 sequence. Blue arrows show amino acids (H83, K86, G87, Q102, and Q105) crucial for functional folding of the protein. (C) Amplified cDNA products of the differentially accumulated YPD1 isoforms (T0 or T1) under different growth conditions (dark vs. light). Size of the cDNA or a fragment from the 100 bp DNA ladder (M) is mentioned. (D) Graphical and qualitative (inset) representation of the ratiometric analysis of the transcript levels of the YPD1 isoforms (T0 and T1) under photo-illumination, with respect to that of tubulin (Tub) as an internal control, in the WT M. oryzae. Arrowhead depicts 500 bp fragment from the 1 Kb DNA ladder (M). The asterisks denote 10-digit interval in the multiple sequence alignment.

Figure 2

Immunolocalization of Ypd1 in M. oryzae. Immunostaining of the fungal samples representing asexual development (conidiophores, upper panels; and mature conidia, middle panels) or pathogenic differentiation (germinating conidia, lower panels). Samples were co-stained with DAPI to mark the nuclei, and anti-Ypd1 antibody followed by TRITC-labeled secondary antibody. Arrowheads denote cytoplasmic signal whereas arrows show punctate signal. Scale bar, 10 μm.

Figure 3

Down-regulation of YPD1 and its downstream effects in M. oryzae. (A) A schematic representation of the genomic locus of the YPD1 open reading frame. The horizontal blue bar denotes the stretch of nucleotide sequence used to make a YPD1 KD construct. (B) A bar graph representing relative YPD1 transcript levels in the indicated KD transformants. Data represent mean ± s.e.m. from three independent experiments. ^**P < 0.01. (C) Western blot analyses showing relative expression level of unphosphorylated or phosphorylated Hog1 protein detected using the relevant indicated antibodies. The blots were stained with Ponceau S to confirm normalization of protein samples. (D) Relative transcript levels of the indicated genes, between the WT or KD transformant RA6, are depicted in a bar graph. Data represent mean ± s.e.m. from three independent assays performed using total RNA from vegetative cultures of the indicated strains of M. oryzae. (E) A bar graph showing relative transcript levels of the indicated PAS-domain HKs in the WT or RA6 KD transformant grown under light or dark condition. Data represents mean ± s.e.m. from three individual experiments.

Figure 4

Ypd1 plays a crucial role in asexual development and pathogenesis in M. oryzae. (A) Top panels show top view of the radial vegetative growth of the indicated M. oryzae strains after 10 days on PA medium. Transverse sections (TS) of the vegetative colonies showing aerial hyphal growth in the WT or KD transformants. Arrow depicts fluffy or thin aerial growth in the WT or the KD transformants, respectively. (B) A bar graph depicting total conidiation in the indicated strains of M. oryzae. Total conidia were harvested from 10-day old cultures of the strains grown on PA medium. Data represents mean ± s.e.m. from three independent experiments. (C) Micrographs showing appressorial development, on an inductive hydrophobic surface 24 hpi, by the indicated strain. Black and white arrowheads depict aberrant-shaped and non-melanized appressoria, respectively. Values indicate percentage (mean ± s.e.m. from three replicates) of appressorial development after 24 hpi. Scale bar, 10 μm. (D) Rice sheath was inoculated with the indicated strain of M. oryzae, and observed under bright field optics after 48 hpi. Asterisk shows appressoria on the sheath tissue surface. Black and white arrowheads depict elaborated or restricted fungal invasive hyphae, respectively, of the WT or YPD1 KD transformant in M. oryzae. Scale bar, 10 μm. (E) Detached rice leaf blades were inoculated with the WT or the YPD1 KD transformant conidial suspension, and the disease symptoms were assessed and photographed 7–8 days post inoculation.