PLS1, a gene encoding a tetraspanin-like protein, is required for penetration of rice leaf by the fungal pathogen Magnaporthe grisea

Abstract

We describe in this study punchless, a nonpathogenic mutant from the rice blast fungus M. grisea, obtained by plasmid-mediated insertional mutagenesis. As do most fungal plant pathogens, M. grisea differentiates an infection structure specialized for host penetration called the appressorium. We show that punchless differentiates appressoria that fail to breach either the leaf epidermis or artificial membranes such as cellophane. Cytological analysis of punchless appressoria shows that they have a cellular structure, turgor, and glycogen content similar to those of wild type before penetration, but that they are unable to differentiate penetration pegs. The inactivated gene, PLS1, encodes a putative integral membrane protein of 225 aa (Pls1p). A functional Pls1p-green fluorescent protein fusion protein was detected only in appressoria and was localized in plasma membranes and vacuoles. Pls1p is structurally related to the tetraspanin family. In animals, these proteins are components of membrane signaling complexes controlling cell differentiation, motility, and adhesion. We conclude that PLS1 controls an appressorial function essential for the penetration of the fungus into host leaves.

Figure 1

Penetration of host leaves by the rice blast fungus. (A) Conidia are disseminated in water drops splashing from a sporulating lesion. (B) A strong glue sticks conidia to the host surface. (C) Appressorium differentiates rapidly after conidium germination. (D) Mature appressorium is a melanized, dome-shaped and thick-walled cell. (E) Penetration peg breaches the plant cuticle and cell wall by mechanical force. After penetration, a bulbous infection hypha invades the epidermal cell. Abbreviations: ml, melanin layer; sp, septum; pp, penetration peg; rg, ring of conidial glue; bih, bulbous infection hypha; pc, plant cuticle; pcw, plant cell wall.

Figure 2

punchless phenotypic analysis. (A) Infection assay on rice. Left, punchless mutant; right, P1.2 wild type. Typical lesions were observed for wild type 7 days after inoculation. (B and C) Sections of barley epidermal cells 20 h after inoculation with P1.2 (B) or punchless (C). A penetration peg (pp) and an infectious hypha are visible below the wild-type appressorium (ap). (Bar = 5 μm.) (D and E) Stained sections of barley leaves 20 h after inoculation with P1.2 (D) or punchless (E). Glycogen is detected only in punchless appressorium as shown by a red staining. (Bar = 5 μm.) (F and G) Barley epidermal strips 2 days after inoculation with P1.2 (F) or punchless (G). Bulbous infection hyphae (ih) are visible under the wild-type appressorium (ap). (Bar = 20 μm.) (H and I) Penetration of cellophane by P1.2. (H) or punchless (I). Five days after inoculation, pseudoinfection hyphae are visible inside the cellophane under the wild-type appressorium. (Bar = 20 μm.)

Figure 3

Cloning of pAN7.1 insertion locus from punchless. (A) punchless genomic DNA gel blot analysis. A single pAN7.1-hybridizing fragment was detected for BglII, which normally cuts once in pAN7.1. SspI hybridization pattern revealed a 4-kb fragment corresponding to a pAN7.1 internal fragment. Analysis of double-digest hybridization patterns showed that the origin of replication and a segment of the ampicillin-resistance gene of pAN7.1 were deleted during its integration. (B) Restriction map of the pAN7.1 insertion locus cloned by inverse PCR. Shaded box, M. grisea genomic DNA. Line and empty box, plasmid DNA. Abbreviations for restriction sites are E, EcoRI; B, BglII; H, HindIII; Sp, SspI; and SacI, Sc. 421(+) and 421(−) are the positions of primers used for inverse PCR. (C) Restriction map of wild-type genomic fragments (15 kb and 8.5 kb) from cosmid 32H7 used for complementation. The 8.5-kb BamHI fragment was cloned in pCB1265 to give plasmid pCM421-8.5. The StuI–ClaI fragment of 1 kb was used as a probe to identify PLS1 cDNA clones.

Figure 4

Predicted secondary structure of Pls1p. (A) Prediction of Pls1p transmembrane domains. Hydropathy plot using the Kyte and Doolittle algorithm. Bars, putative transmembrane domains (TM1, -2, -3, and -4). (B) Predicted secondary structure of Pls1p. Cysteine-based patterns conserved among tetraspanins are CCG at position 137, CP at position 154, and GC at position 165. Charged amino acids located in transmembrane domains are at positions 13 (D), 61 (N), 94 (N), and 192 (D). Transmembrane domains are indicated as open cylinders. (C) Cysteine-based patterns in ECL2 of Pls1p and animal tetraspanins. The distance between each conserved cysteine-based pattern is given in aa. Variations of the XCPK/S pattern in tetraspanins CD9 and CD81 are given under Pls1p pattern.

Figure 5

Expression and localization of Pls1p in the appressorium during infection of barley leaves. A transformant expressing a Pls1p-GFP fusion under the control of the PLS1 promoter and terminator was inoculated to detached barley leaves and observed by confocal laser scanning microscopy. Two classes of appressoria were observed on the leaf surface 12–14 h after inoculation. (A) GFP fluorescence of class I appressorium. (Bar = 4 μm.) A faint fluorescence was detected at the appressorium periphery. (B) Same view as in A under bright light. (C) GFP fluorescence of class II appressorium. A strong fluorescence was detected in vacuoles and a faint fluorescence was detected at the appressorium periphery. (Bar = 4 μm.) (D) Same view as in C under bright light. Most appressoria (80%) observed belonged to class II.