ER retention receptor, MoERR1 is required for fungal development and pathogenicity in the rice blast fungus, Magnaporthe oryzae

Abstract

ER retention receptor is a seven trans-membrane protein that plays pivotal roles in function and integrity of endoplasmic reticulum (ER). Insertional mutagenesis of Magnaporthe oryzae identified MoERR1 as a pathogenicity gene encoding putative ER retention receptor orthologous to ERD2 in Saccharomyces cerevisiae. Search through the genome identified that M. oryzae possesses another ortholog of ERD2, which is designated as MoERR2. When MoERR1 and MoERR2 were tagged with GFP, both were localized to ER. Targeted disruption of MoERR1 showed pleiotropic effects on phenotypes, while deletion of MoERR2 had no effect on phenotypes we examined. The disruption mutant of MoERR1 showed growth retardation and produced significantly reduced number of conidia with aberrant morphology. Appressoria from the mutant were unable to penetrate into plant tissues presumably due to defect in cell wall integrity, thereby rendering the mutant non-pathogenic. The MoERR1 mutant also appeared to display abnormal ER structure and mis-regulation of genes involved in chaperone function and unfolded protein response under ER stress condition. Taken together, these results suggest that MoERR1 is a ER retention receptor required for function and integrity of ER, and that MoERR1-mediated ER functionalities are essential for fungal development and pathogenesis.

Figure 1

Identification of T-DNA insertion in MoERR1 ^T-DNA and hydropathy plot for MoERR1, MoERR2, and ERD2. (A) DNA sequences of genome in and around T-DNA insertions sites. Upper cases represent left and right border sequences of T-DNA and lower cases genomic sequences. (B) Hydropathy plot MoERR1, MoERR2, and ERD2 (from top to bottom). Transmembrane regions were predicted by DAS server (http://www.sbc.su.se/~miklos/DAS/). Solid and dotted lines indicate strict (2.2) and loose threshold score (1.7), respectively. Arrows indicate transmembrane regions predicted with loose threshold.

Figure 2

Targeted gene disruption of MoERR1 and MoERR2. (A) MoERR1 gene disruption strategy. Moerr1 ^T-DNA was generated by targeted same allele of knock-out construct obtained from MoERR1 ^T-DNA. (B) Southern hybridization of MoERR1 mutants. Genomic DNA was digested by HindIII, and probed by 3′ flanking 1 kb fragment. Lane 1: wild type strain KJ201; Lane 2: MoERR1 ^T-DNA; Lane 3: Moerr1 ^T-DNA; Lane 4: E1-46 (ectopic transformant). Blot image were cropped for better display. (C) MoERR2 gene deletion strategy using double-joint PCR. Knockout construct was designed to include upstream ~1 kb fragment and downstream ~1 kb fragment of MoERR2 ORF linked with HPH cassette. (D) Southern hybridization of MoERR2 mutants. Genomic DNA was digested by SalI, and probed by 5′ flanking 1 kb fragment. Lane 1: wild type strain KJ201; Lane 2: E1-3 (ectopic transformant); Lane 3: ∆Moerr2; Lane 4: E1-46 (ectopic transformant). Blot image was cropped for better display.

Figure 3

Cellular localization of MoERR1::eGFP and MoERR2::eGFP. MoERR1 or MoERR2 eGFP tagging construct with 1 kb of native promoter region was introduced in wild type strain KJ201. Conidia of GFP tagging strains were stained with 10 mM of the ER-Tracker dye Blue-White DPX, and observed using a 4′,6-diamidino-2-phenylindole filter after incubation for 30 minutes. Blue color of conidia stained by ER tracker was converted to red for better visualization of co-localization. Bar = 10 μm.

Figure 4

Conidiation and conidiophore development. (A) Conidiation was quantified at 10 dpi on oatmeal agar. The values are the means with SD of three replicates. (B) Conidiophore development was observed under light microscope at 18 hpi. (C) Conidiophore was examined by scanning electron microscopy. Magnification of upper panel was x750 and that of lower panel was x2000.

Figure 5

Conidia morphology of Moerr1 ^T-DNA. (A) Distribution of conidia type by cell number. Each ratio of conidia types were calculated with more than 100 conidia examined by 3 replications. (B) Cell morphology. Conidial cell wall were stained by calcofluore white, and observed using a 4′,6-diamidino-2-phenylindole filter after incubation for 10 minutes. Bar = 10 μm.

Figure 6

Pathogenicity of wild type and MoERR1 mutants. (A) Spray inoculation. Disease symptoms on rice leaves were examined at 7 dpi on susceptible rice cv. Nakdong. (B) Infiltration inoculation. Disease symptoms were examined at 7 dpi on artificial wound. (C) Rice sheath infection at 48 hpi. Fungal invasive growth in rice sheath cell was observed under light microscopy. Arrows indicate appressorium. Bar = 20 μm.

Figure 7

Cellular localization of eGFP-HDEL and eGFP-HEEL in conidia. GFP with ER retention signal construct was designed to have GFP tagging with HDEL/HEEL in N-terminal and signal peptide of LHS1 or KAR2 in C-terminal. Bar = 10 μm.

Figure 8

Gene expression of MoERR1, MoERR2, KAR2 and LHS1 under ER stress condition. Mycelia were grown on liquid complete medium for 3 days, and transferred to new medium. After incubation of 1 day, 10 mM DTT treatment for 30 minutes was used as ER stress condition. Partial cDNA fragment of MoERR1, MoERR2, KAR2 and LHS1 were used as probes of northern blot analysis. Blot and gel images were cropped for better display.