Different chitin synthase genes are required for various developmental and plant infection processes in the rice blast fungus Magnaporthe oryzae

Abstract

Chitin is a major component of fungal cell wall and is synthesized by chitin synthases (Chs). Plant pathogenic fungi normally have multiple chitin synthase genes. To determine their roles in development and pathogenesis, we functionally characterized all seven CHS genes in Magnaporthe oryzae. Three of them, CHS1, CHS6, and CHS7, were found to be important for plant infection. While the chs6 mutant was non-pathogenic, the chs1 and chs7 mutants were significantly reduced in virulence. CHS1 plays a specific role in conidiogenesis, an essential step for natural infection cycle. Most of chs1 conidia had no septum and spore tip mucilage. The chs6 mutant was reduced in hyphal growth and conidiation. It failed to penetrate and grow invasively in plant cells. The two MMD-containing chitin synthase genes, CHS5 and CHS6, have a similar expression pattern. Although deletion of CHS5 had no detectable phenotype, the chs5 chs6 double mutant had more severe defects than the chs6 mutant, indicating that they may have overlapping functions in maintaining polarized growth in vegetative and invasive hyphae. Unlike the other CHS genes, CHS7 has a unique function in appressorium formation. Although it was blocked in appressorium formation by germ tubes on artificial hydrophobic surfaces, the chs7 mutant still produced melanized appressoria by hyphal tips or on plant surfaces, indicating that chitin synthase genes have distinct impacts on appressorium formation by hyphal tip and germ tube. The chs7 mutant also was defective in appressorium penetration and invasive growth. Overall, our results indicate that individual CHS genes play diverse roles in hyphal growth, conidiogenesis, appressorium development, and pathogenesis in M. oryzae, and provided potential new leads in the control of this devastating pathogen by targeting specific chitin synthases.

Figure 1. The seven predicted CHS genes in M. oryzae.

(A) Domain structures of the seven chitin synthases in M. oryzae. Chs, chitin synthase domain; Cyt-b5, cytochrome b5-like heme/steroid binding domain; MMD, myosin motor domain; TM, transmembrane domain. (B) Directions and chromosomal positions of CHS5 and CHS6 in M. oryzae and their orthologs in F. graminearum, A. fumigatus, and N. crassa. RBS, Rlm1p-binding sequence.

Figure 2. Expression profiles of seven CHS genes assayed by qRT-PCR.

RNA samples of the wild-type strain P131 were isolated from vegetative hyphae grown in minimal medium (VH), conidia harvested from 10-day-old oatmeal agar cultures (CO), 24 h appressoria (AP), and infected plant leaves (IP). The relative expression level of individual CHS genes was analyzed with the 2^−ΔΔCt method with the actin gene as the internal control for normalization. (A) Comparison of the transcript abundance of seven CHS genes in VH, CO, AP, and IP. The expression level of CHS1 was arbitrarily set to 1. Columns 1 to 7 represent the chitin synthase genes CHS1 to CHS7. (B) Comparison of the transcript abundance of individual CHS genes in four different fungal tissues. The expression level of each CHS gene in vegetative hyphae (VH) was arbitrarily set to 1. Mean and standard errors were determined with data from three independent replicates.

Figure 3. Colony morphology and melanized appressoria of the chs deletion mutants.

(A) Colonies of the wild-type strains P131 and S1528 and the chs1-chs7 mutants formed on the complete medium (CM) at 25°C. Photos were taken at 7 days post-inoculation. Bar = 10 mm. (B) Appressorium formation assays with conidia from the same set of the wild-type and mutant strains. Representative images were taken after 24 h incubation on glass coverslips. Germ tubes of the chs7 mutant failed to form appressoria. Bar = 10 µm.

Figure 4. Chitin contents and chitin synthase activities in the wild-type and chs mutant strains.

The chitin content was assayed with vegetative hyphae harvested from 2-day-old CM cultures (A) or conidia harvested from 10-day-old OTA plates (B) of the wild type and chs mutants. The chitin content is expressed in µg of glucosamine hydrochloride per mg of dry-weight of fungal biomass. (C) Chitin synthase activities (nM UDP-N-acetyl-glucosamine incorporated into chitin per mg protein per minute) were assayed with microsomal fractions of proteins isolated from vegetative hyphae of the wild-type and chs mutant strains. Mean and standard deviation were calculated with results from three biological replicates.

Figure 5. The expression levels of individual CHS genes in the wild type and chs mutants.

RNA samples isolated from vegetative hyphae harvested from 2-day-old CM cultures were used for qRT-PCR assays. The actin gene was used as the endogenous control for normalization. Relative expression levels were estimated with the 2^−ΔΔCt method. The expression level of each CHS gene in the wild type was arbitrarily set to 1. Mean and standard errors were determined with data from three independent replicates.

Figure 6. Appressorium formation assays with the chs7 mutant.

Melanized appressoria were formed by the germ tubes of the wild-type strain P131 and chs7 mutant on onion epidermis cells, rice leaf sheath, and barley leaves. AP, appressoria formed by germ tubes; CO, conidia. Bar = 10 µm.

Figure 7. Appressorium formation assays with hyphal tips.

(A) Appressoria formed by hyphal tips of the wild-type strain P131 and chs1-chs7 mutants on glass cover slips. Bar = 10 µm. (B) Appressoria formed by hyphal tips of P131 and chs7 mutant on barley leaf and rice root surfaces. AP, appressoria formed by hyphal tips; HY, hyphae. Bar = 10 µm.

Figure 8. Infection assays with rice and barley seedlings.

Conidia were harvested from 10-day-old oatmeal agar cultures of wild-type strains P131 and S1528, and the chs1-chs7 mutants. (A) Eight-day-old seedlings of barley cultivar Golden Promise were spray inoculated. Typical leaves were photographed 5 dpi. (B) Two-week-old seedlings of rice cultivar LTH were sprayed with conidium suspensions of the wild-type and chs mutant strains or 0.25% gelatin solution as the control. Photos were taken at 7 dpi.

Figure 9. The chs7 mutant was reduced in virulence.

(A) Eight-day-old barley leaves were drop-inoculated with conidium suspensions of the wild type and chs7 mutant. The concentration of each conidium suspensions was marked to the left. Inoculation with 0.25% gelatin was used as the control. Typical leaves were photographed 5 dpi. (B) Rice sheaths and barley leaves were drop-inoculated with conidia of the wild type and chs7 mutant. Penetration and invasive hyphae were examined 48 hpi. AP, appressoria; IH, infection hyphae. Bar = 10 µm.

Figure 10. CHS1 is important for conidiogenesis.

(A) Typical conidia of the wild-type strain P131 and chs1 mutant were examined under differential interference contrast (DIC) microscope. (B) The percentage of conidia with 0-, 1-, and 2-septa in the wild-type strain P131 and chs1 mutant. The chs1 mutant failed to produce normal, three-celled pyriform conidia. (C) Spore tip mucilage of the conidia of the wild type and chs1 mutant were stained with Calcofluor White (CFW) and examined by DIC and epifluorescence (UV) microscopy. Arrows pointed to STM. Bar = 10 µm. (D) Conidia and conidiophores of the wild-type P131 and chs1 mutant examined under scanning electron microscope (SEM). CO, conidium; CP, conidiophores; STM spore tip mucilage. Bar = 10 µm.

Figure 11. The chs1 mutant was defective in appressorium penetration and plant infection.

(A) Appressorium penetration assays with rice leaf sheaths. Appressoria of the chs1 mutant failed to penetrate plant cells and develop infectious hyphae at 48 hpi. (B) Intact and wounded barley leaves were inoculated with culture blocks of the wild-type P131 and chs1 mutant. Lesions were visible 5 dpi after removing the inoculum. Inoculation with water agar blocks was the negative control. AP, appressorium; IH, invasive hyphae; PP, penetration peg.

Figure 12. Chs1-eGFP fusion proteins accumulate to the tips.

(A) Conidia produced by transformant LA33 expressing the CHS1-eGFP construct were observed by DIC and epifluorescence microscopy. (B) Hyphal tips and developing conidia of transformant LA33. The Chs1-eGFP fusion protein localized to the tip region in vegetative hyphae (the upper panel) and developing a young conidium (the bottom panel) at the tip of a conidiophore. CO, conidia; CP, conidiophores; HT, hyphal tips; HY, hyphae; YC, young conidia. Bar = 10 µm.

Figure 13. The chs6 mutant and chs5 chs6 double mutant failed to penetrate the plant cell.

(A) Appressorium penetration assays with onion epidermal cells. Appressoria of the chs6 mutant and chs5 chs6 double mutant failed to penetrate plant cells and develop infectious hyphae at 48 hpi. Bar = 10 µm. (B) ROS accumulation in the infected barley leaves by the chs6 mutant stained with DAB. Strong ROS accumulations were observed in the leaves inoculated with the chs6 mutant. Bar = 10 µm. (C) Wounded barley leaves were inoculated with culture blocks of the wild-type P131, chs5, chs6, and chs5 ch6 double mutant. Lesions were visible 5 dpi after removing the inocula. Inoculation with water agar blocks was the negative control. (D) Vegetative hyphae of the wild-type P131 and the chs5, chs6, and chs5 chs6 mutants from 2-day-old 5×YEG cultures. Bar = 10 µm. AP, appressorium; IH, invasive hyphae; PP, penetration peg.