ShORR-1, a Novel Tomato Gene, Confers Enhanced Host Resistance to Oidium neolycopersici

Abstract

A previous complementary cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis examined responses to the powdery mildew pathogen Oidium neolycopersici (On) of the resistant cultivar Solanum habrochiates G1.1560, carrying the Ol-1 resistance gene, and susceptible cultivar S. lycopersicum Moneymaker (MM). Among other findings, a differentially expressed transcript-derived fragment (DE-TDF) (M14E72-213) was upregulated in near isogenic line (NIL)-Ol-1, but absent in MM. This DE-TDF showed high homology to a gene of unknown function, which we named ShORR-1 (Solanum habrochaites Oidium Resistance Required-1). However, MM homolog of ShORR-1 (named ShORR-1-M) was still found with 95.26% nucleic acid sequence similarity to ShORR-1 from G1.1560 (named ShORR-1-G); this was because the cut sites of restriction enzymes in the previous complementary cDNA-AFLP analysis was absent in ShORR-1-M and differs at 13 amino acids from ShORR-1-G. Transient expression in onion epidermal cells showed that ShORR-1 is a membrane-localized protein. Virus-induced gene silencing (VIGS) of ShORR-1-G in G1.1560 plants increased susceptibility to On. Furthermore, overexpressing of ShORR-1-G conferred MM with resistance to On, involving extensive hydrogen peroxide accumulation and formation of abnormal haustoria. Knockdown of ShORR-1-M in MM did not affect its susceptibility to On, while overexpressing of ShORR-1-M enhanced MM's susceptibility to On. We also found that changes in transcript levels of six well-known hormone signaling and defense-related genes are involved in ShORR-1-G-mediated resistance to On. The results indicate that ShORR-1-M and ShORR-1-G have antagonistic effects in tomato responses to On, and that ShORR-1 is essential for Ol-1-mediated resistance in tomato.

Keywords: H2O2 accumulation; Oidium neolycopersici; Ol-1-mediated resistance; abnormal haustoria; resistant tomato; susceptible tomato.

Figure 1

Nucleotide (A) and amino acid (B) sequences alignment of ShORR-1-M and ShORR-1-G share high homology using DNAMAN software.

Figure 2

ShORR-1-M and ShORR-1-G both localized to the plasma membrane. Subcellular localization GFP fusions with ShORR-1 variants isolated from G1.1560 and MM in onion epidermal cells, as shown by 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining (A, E, and I), green fluorescent protein (GFP) fluorescence (B, F, and J), merger of DAPI staining and fluorescence (C, G, and K), bright field microscopy (D, H, and L). The proteins displayed in panels (A–D), (E–H), and (I–L) were transiently expressed using pSAT6-GFP-N1-ShORR-1-G, pSAT6-GFP-N1-ShORR-1-M, and a construct designed to express GFP alone (as a control), respectively. Scale bar = 25 µm.

Figure 3

Expression profiles of ShORR-1 at indicated On infection stages and tissues in MM (A) and G1.1560 (B). Samples were harvested at 0, 8, 24, 36, 72, and 120 hpi (A–B). The relative expression levels of ShORR-1 at each time-point were normalized relative to SlActin. Asterisks indicate significant difference from the control determined by one-way ANOVA followed by an independent-samples Dunnett’s post hoc test (**: P < 0.01). (C) The transcript accumulation of ShORR-1 was examined by quantitative real-time PCR from MM tissues, including roots (R), stems (S), leaves (L), flowers (F), green fruits (G), and red fruits (M). Actin was used as an internal control. Mean and standard error were calculated using data from three independent biological replicates. Letters indicate significant differences between different tissues determined by one-way ANOVA followed by Tukey’s HSD test (P < 0.01).

Figure 4

Downregulation of ShORR-1 by VIGS compromises Ol-1-mediated resistance. (A) On susceptible phenotypes of MM and ShORR-1-silenced (TRV2-ShORR-1) G1.1560 plants, and resistant phenotype of G1.1560 controls transformed with an empty vector (TRV2-EV). (B) Levels of ShORR-1 transcripts in control and TRV2-ShORR-1 leaves. Error bars represent standard deviations, obtained from analysis of three biological replicates. Asterisks indicate significant difference from the control by one-way ANOVA followed by an independent-samples Dunnett’s post hoc test (**: P < 0.01). (C) Microscopic morphologies of On in control and TRV2-ShORR-1 plants. Bar = 25 µm.

Figure 5

ShORR-1-silenced plants show decreased On-induced HR formation and H₂O₂ accumulation. (A) Micrographs of resistant S. habrochaites G1.1560 without ShORR-1 silencing (left) and ShORR-1-silenced S. habrochaites G1.1560 (right). Bar = 15 µm. (B) Percentages of HR-associated primary and secondary haustoria (HS) in ShORR-1-silenced and G1.1560 control plants. Asterisks indicate significant difference from the control by one-way ANOVA followed by an independent-samples Dunnett’s post hoc test (**: P < 0.01, *: P < 0.05). Three biological replicates of microscopic samples of both ShORR-1 silenced and control plants were observed.

Figure 6

Overexpressing of ShORR-1-G enhanced tomato plants’ resistance to On. (A) Micrographs of powdery mildew on leaves of T2 ShORR-1-G overexpressing plants and untransformed MM plants, 65 h after inoculation. Scale bar = 25 µm. (B) Macroscopic phenotypes of On infected leaves of untransformed MM plants and T2 ShORR-1-G overexpressing plants. (C) Percentages of germinated On conidiospores on untransformed MM plants and T2 ShORR-1-G overexpressing plants at 65 h after infection. (D) Levels of ShORR-1 transcripts in three transformed plants and controls. Double asterisks indicate significant differences from the control by one-way ANOVA followed by an independent-samples Dunnett’s post hoc test (P < 0.01). (E) Estimated On fungal biomass on control plants and three transformed lines. All the above results are based on analyses of three biological replicates of control and transgenic plants.

Figure 7

Overexpressing of ShORR-1-M increased susceptibility to On, but silencing it did not increase resistance to On. (A) Micrographs of powdery mildew on leaves of T2 ShORR-1-M overexpressing and silenced plants, compared with untransformed MM plants, 65 h after inoculation. Scale bar = 25 µm. (B) Macroscopic phenotypes of On infected leaves of T2 ShORR-1-M overexpressing and silenced plants. (C) Percentages of germinated On conidiospores on ShORR-1-M overexpressing and silenced plants, 65 h after infection. (D) Levels of ShORR-1 transcripts in transgenic plants and controls. Double asterisks indicate significant differences from the control by one-way ANOVA followed by an independent-samples Dunnett’s post hoc test (P < 0.01). (E) Estimated On fungal biomass on transgenic and control plants. All the above results are based on analyses of three biological replicates of control and transgenic plants.

Figure 8

Overexpressing of ShORR-1-G increased H₂O₂ accumulation in transgenic tomatoes. (A) Wild-type tomato leaves were inoculated with On and sampled 65 hours after infection. (B) Enlarged view of A. (C) ShORR-1-G overexpressing tomato leaves challenged with On and sampled 65 h after infection. (D) Enlarged view of (C), showing H₂O₂ accumulation in epidermal cells, as manifested by 3,3-diaminobenzidine (DAB) staining. Scale bars = 25 µm in (A and C), 12.5 µm in (B and D).

Figure 9

Micrographs of haustorium phenotypes in ShORR-1-G overexpressing plants. Red and black arrows show morphologically abnormal and normal haustoria with high H₂O₂ contents, respectively. Scale bar = 12.5 µm. Panels A–D show the different haustorium phenotypes in ShORR-1-G overexpressing lines 5, 7, 12 and 13.

Figure 10

Quantitative real-time PCR analysis of expression levels of the following selected defense marker genes in wild-type and ShORR-1-G overexpressing (OE) transgenic tomatoes with and without On infection: (A) SlPR1, (B) SlPR2, (C) SlCOI1, (D) SlHSR203J, (E) SlROR2, (F) SlBI1. Actin was used as an internal control. Value for each sample is mean of three biological replicates. Mean and standard error were calculated using data from three independent biological replicates. Lowercase letters were used to indicate significant differences between lines in untreated (without On infection) conditions, and capital ones indicate significant differences between lines after On infection. Upper and lowercase letters indicate significant differences relative to control (WT) plants determined by one-way ANOVA followed by Tukey’s HSD test (P < 0.05).