Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans

Abstract

Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans. Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans. Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H(2)O. Uptake studies with (14)C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, (14)C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.

Figure 1

Morphology of Phytophthora infestans germinating cysts treated with mandipropamid (MPD). (A) Germinating cysts after 3 h without MPD treatment produce long germ tubes of uniform diameter. (B) Germinating cysts after 3 h in 68 nm MPD produce germ tubes with swollen tips. (C) Germinating cysts treated with 68 nm MPD for 1 h, washed three times with H₂O and allowed to grow for a further 2 h on a glass slide show resumed normal growth from the previously swollen germ tube tips. Scale bars in all images represent 125 µm.

Figure 2

Effect of mandipropamid (MPD) on cellulose synthesis and germ tube elongation. Effect of MPD on the uptake of D‐(U‐¹⁴C)‐glucose into Phytophthora infestans germinating cysts (black bars) and incorporation of radiolabel into cellulose (grey bars). Germ tube growth was evaluated in aliquots of each treatment (hatched bars). The percentage uptake represents the percentage of added radioactivity taken up by the cells. The percentage incorporation is the percentage of radioactivity taken up by the culture, which has been incorporated into cellulose. Data are the means ± SE (n= 3).

Figure 3

Sensitivity of wild‐type (wt) and mutated Phytophthora infestans to mandipropamid (MPD). Sensitivity was assessed by growth inhibition on rye agar containing increasing concentrations (µm) of MPD. The minimum inhibitory dose determined for isolate T30‐4 was 0.07 µm. The maximum concentration shown here (35 µm) represents 500 × the minimum inhibitory dose. The growth of mutant Tmut3 was unaffected by any concentration tested, whereas mutant Tmut7 exhibited reduced growth at higher MPD doses. Mutant Tmut7 showed slower growth compared with the wild‐type on unamended rye agar.

Figure 4

Structures of oospores originating from sexual crosses between wild‐type A2 isolate 88133 and Tmut3. Oospores formed in situ in mating cultures of Phytophthora infestans wild‐type A2 testers with wild‐type A1 T30‐4 or mandipropamid (MPD)‐insensitive mutants. (A, B) Oospores from mating cultures of T30‐4 and 88133. Oospores are typically abundant, thick‐walled and with cytoplasm filling the oosphere. (C–E) Oospores from mating cultures of Tmut3 and 88133. Oospores are sparsely distributed, irregular (E) or thin‐walled (D), and cytoplasm is condensed/granular or absent (C). Scale bars in all images represent 20 µm.

Figure 5

Structure and site of mutations to mandipropamid (MPD) insensitivity in the PiCesA3 gene. (A) Intron/exon structure of the PiCesA3 gene. Numbers correspond to the size in base pairs. Point mutations in Tmut3 and Tmut7 and the predicted amino acid exchanges in the mutant gene products are indicated. (B) Alignment of the amino acid sequence of CesA1, CesA2, CesA3 and CesA4 between Phytophthora infestans, P. ramorum and P. sojae at the site of mutation to MPD insensitivity. Gly1105 is conserved in each of the four CesA genes.

Figure 6

Sensitivity of wild‐type and transformed Phytophthora infestans to mandipropamid (MPD). Sensitivity was assessed by growth inhibition on rye agar containing increasing concentrations of MPD. Growth is expressed as millimetres of radial expansion. The minimum inhibitory dose determined for all isolates was 0.07 µm. (A) Transformants with the PiCesA3 Tmut3 mutated allele were assessed for sensitivity to MPD. The maximum concentration at which growth was recorded (2.5 µm) represents 35.7 × the minimum inhibitory dose, whereas the maximum MPD concentration tested here (25 µm) represents 357 × the minimum inhibitory dose. Transformants CES 3‐1 and CES 3‐4 showed clear shifts in MPD sensitivity. (B) Transformants with the wild‐type PiCesA3 allele were assessed for sensitivity to MPD. The maximum MPD concentration is 0.7 µm and no transformant expressing the wild‐type PiCesA3 allele grew at this concentration. Black bars, MPD‐free medium; white bars, 0.07 µm MPD; grey bars, 0.175 µm MPD; checkered bars, 0.35 µm MPD; hatched bars, 0.7 µm MPD; crossed bars, 2.5 µm MPD.