Oxaloacetate acetylhydrolase gene mutants of Sclerotinia sclerotiorum do not accumulate oxalic acid, but do produce limited lesions on host plants

The oxaloacetate acetylhydrolase (OAH, EC 3.7.1.1)‐encoding gene Ss‐oah1 was cloned and functionally characterized from Sclerotinia sclerotiorum. Ss‐oah1 transcript accumulation mirrored oxalic acid (OA) accumulation with neutral pH induction dependent on the pH‐responsive transcriptional regulator...

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Published in:	Molecular plant pathology Vol. 16; no. 6; pp. 559 - 571
Main Authors:	Liang, Xiaofei, Liberti, Daniele, Li, Moyi, Kim, Young‐Tae, Hutchens, Andrew, Wilson, Ron, Rollins, Jeffrey A
Format:	Journal Article
Language:	English
Published:	England Blackwell Science in collaboration with the British Society of Plant Pathology 01-08-2015 Blackwell Publishing Ltd John Wiley & Sons, Inc John Wiley and Sons Inc
Subjects:	appressoria Ascomycota - enzymology Ascomycota - genetics Ascomycota - metabolism Ascomycota - pathogenicity biogenesis Cloning, Molecular gene deletion genes host plants Hydrolases - genetics hyphae mutants Mutation necrotroph Original oxalate oxalic acid Oxalic Acid - metabolism pathogenesis phytopathogen RNA, Messenger - genetics Sclerotinia Sclerotinia sclerotiorum transcription factors viability Virulence pathogenesis virulence necrotroph phytopathogen oxalate Sclerotinia oxalic acid
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Summary:	The oxaloacetate acetylhydrolase (OAH, EC 3.7.1.1)‐encoding gene Ss‐oah1 was cloned and functionally characterized from Sclerotinia sclerotiorum. Ss‐oah1 transcript accumulation mirrored oxalic acid (OA) accumulation with neutral pH induction dependent on the pH‐responsive transcriptional regulator Ss‐Pac1. Unlike previously characterized ultraviolet (UV)‐induced oxalate‐deficient mutants (‘A’ mutants) which retain the capacity to accumulate OA, gene deletion Δss‐oah1 mutants did not accumulate OA in culture or during plant infection. This defect in OA accumulation was fully restored on reintroduction of the wild‐type (WT) Ss‐oah1 gene. The Δss‐oah1 mutants were also deficient in compound appressorium and sclerotium development and exhibited a severe radial growth defect on medium buffered at neutral pH. On a variety of plant hosts, the Δss‐oah1 mutants established very restricted lesions in which the infectious hyphae gradually lost viability. Cytological comparisons of WT and Δss‐oah1 infections revealed low and no OA accumulation, respectively, in subcuticular hyphae. Both WT and mutant hyphae exhibited a transient association with viable host epidermal cells at the infection front. In summary, our experimental data establish a critical requirement for OAH activity in S. sclerotiorum OA biogenesis and pathogenesis, but also suggest that factors independent of OA contribute to the establishment of primary lesions.
Bibliography:	http://dx.doi.org/10.1111/mpp.12211 istex:81706300BB8DCB128BBD9E41E64B91A77BF3DC5D ArticleID:MPP12211 ark:/67375/WNG-C9DLGH3R-8 University of Florida National Sclerotinia Initiative Fig. S1 Generation and verification of Ss-oah1 gene replacement mutants. (A) Schematic representation of the Ss-oah1 genomic locus and expected gene replacement event. (B) Southern blot analysis of the wild-type (WT), knockout mutants (KO1, KO2) and complementation strain (Com). Left panel: HindIII (H)-digested total genomic DNA hybridized with probe 1. Right panel: HindIII (H)- and XbaI (X)-digested total genomic DNA hybridized with probe 2. UTR, untranslated region.Fig. S2 Radial growth kinetics on potato dextrose agar (PDA) medium and PDA medium buffered with citric acid-sodium phosphate buffer at the indicated initial pH values. Data points represent the mean values from three independent colony replicates. The standard deviations for all data points did not exceed 0.3 and were not plotted. WT, wild type; A2, UV-induced oxalate-deficient mutant; KO1 and KO2, Δss-oah1 gene deletion mutants; Com, complementation strain.Fig. S3 'Green island/necronissia' lesions produced by the Δss-oah1 mutant (KO2) on detached soybean (A) and detached canola (B) leaves. Leaves were wounded prior to inoculation. Leaves in (A) were photographed at 5 days post-inoculation. WT, wild-type.Fig. S4 Exogenous oxalic acid (OA) treatment partially restored the virulence defect of the Δss-oah1 mutants (KO1, KO2). Following petiole immersion in OA (pH 5.8) or double-distilled H2O, trifoliate leaves were wounded and inoculated with mycelial plugs (3 days post-inoculation). CK, non-inoculated control.Fig. S5 Cytological events related to the infection of Sclerotinia sclerotiorum on onion epidermal strips sampled between 12 and 24 h post-inoculation (hpi). (A) Compound appressorium-mediated cuticle penetration; arrowhead indicates the differentiation of nascent, bulbous subcuticular hyphae. (B) Papilla deposition and cell wall autofluorescence at the penetration sites. (C) Densely aligned subcuticular hyphae formed at the initial penetration site; arrowhead indicates the sheath layer surrounding subcuticular hyphae. (D) An established infection detailing where infectious hyphae had spread and killed epidermal cells over a large area. (E) A subcuticular hypha showing initial contact with the host plasma membrane (arrowhead); note the loss of plasmolysis activity of the penetrated cell, but not the two neighbouring cells. (F) Subcuticular hyphae penetrating into the anticlinal cell wall (arrowheads). P, penetration sites; PM, plasma membrane; SH, subcuticular hyphae. The scale bars represent 100 μm.Fig. S6 Frequent association between subcuticular hyphae and viable (plasmolysis-positive) onion epidermal cells observed with the wild-type (WT) and the Δss-oah1 mutant (KO2). Plasmolysed onion epidermal cells in association with fungal subcuticular hyphae are marked with red dots. Scale bars represent 200 μm. GFP, green fluorescent protein.Fig. S7 Oxalic acid (OA) crystal accumulation dynamics during onion epidermal peel colonization by Sclerotinia sclerotiorum wild-type (WT). (A, B) Penetration stage OA crystals. Crystals were rarely observed surrounding epiphytic hyphae or compound appressoria on the cuticle surface, but were frequently observed at the penetration points. (C) OA crystals at the early infection stages. Crystals did not accumulate around subcuticular hyphae, but accumulated abundantly within old compound appressoria. (D, E) OA crystals at advanced infection stages. Crystals accumulated heavily within fully disrupted epidermal cells at the lesion centre, but occurred sporadically at the infection front; some crystals accumulated below (on the adaxial surface) viable epidermal cells in areas ahead of the colonization front, which became more abundant as colonization proceeded (dead epidermal cells in E are indicated by a red dot). CA, compound appressoria; P, penetration sites; SH, subcuticular hyphae; VH, vegetative hyphae. Scale bars represent 100 μm.Table S1 PacC binding sites (GCCARG) at the upstream intergenic regions of Ascomycota oah homologues. oah orthologues are defined as those showing gene order synteny conservation with known oah genes. Table S2 Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) quantification of Ss-oah1 gene expression during compound appressorium development. Table S3 Lesion development and oxalic acid (OA) accumulation.
ISSN:	1464-6722 1364-3703
DOI:	10.1111/mpp.12211