Two interacting ethylene response factors negatively regulate peach resistance to Lasiodiplodia theobromae

Abstract

Gummosis is 1 of the most common and destructive diseases threatening global peach (Prunus persica) production. Our previous studies have revealed that ethylene and methyl jasmonate enhance peach susceptibility to Lasiodiplodia theobromae, a virulent pathogen inducing gummosis; however, the underlying molecular mechanisms remain obscure. Here, 2 ethylene response factors (ERFs), PpERF98 and PpERF1, were identified as negative regulators in peach response to L. theobromae infection. Expression of 2 putative paralogs, PpERF98-1/2, was dramatically induced by ethylene and L. theobromae treatments and accumulated highly in the gummosis-sensitive cultivar. Silencing of PpERF98-1/2 increased salicylic acid (SA) content and pathogenesis-related genes PpPR1 and PpPR2 transcripts, conferring peach resistance to L. theobromae, whereas peach and tomato (Solanum lycopersicum) plants overexpressing either of PpERF98-1/2 showed opposite changes. Also, jasmonic acid markedly accumulated in PpERF98-1/2-silenced plants, but reduction in PpPR3, PpPR4, and PpCHI (Chitinase) transcripts indicated a blocked signaling pathway. PpERF98-1 and 2 were further demonstrated to directly bind the promoters of 2 putative paralogous PpERF1 genes and to activate the ERF branch of the jasmonate/ethylene signaling pathway, thus attenuating SA-dependent defenses. The lesion phenotypes of peach seedlings overexpressing PpERF1-1/2 and PpERF98-1/2 were similar. Furthermore, PpERF98-1/2 formed homodimers/heterodimers and interacted with the 2 PpERF1 proteins to amplify the jasmonate/ethylene signaling pathway, as larger lesions were observed in peach plants cooverexpressing PpERF98 with PpERF1 relative to individual PpERF98 overexpression. Overall, our work deciphers an important regulatory network of ethylene-mediated peach susceptibility to L. theobromae based on a PpERF98-PpERF1 transcriptional cascade, which could be utilized as a potential target for genetic engineering to augment protection against L. theobromae-mediated diseases in crops and trees.

Figure 1.

PpERF98-1/2 transcripts accumulate in the infected shoots of a peach cultivar susceptible to L. theobromae. A and B) Morphological progression of gummosis in the current-year shoots and fully expanded leaves inoculated with L. theobromae. All leaf images were digitally extracted with the same scale in panels A (bar = 1 cm) and B (bar = 2 cm). C and D) Comparison of lesion size in the infected shoots and leaves between the susceptible cultivar ‘Spring Snow' (SS) and the tolerant cultivar ‘Da Hongpao' (DHP). Asterisks indicate statistical significance between SS and DHP at the same timepoint with *P < 0.05, **P < 0.01 using Student's t test. E and F) Relative transcript levels of PpERF98-1/2 in infected shoots normalized on the reference gene PpTEF2, and displayed as fold changes compared with mock samples at time 0 h. Hpi: hours post inoculation. Different letters show statistical significance of different treatments at the same time point and P < 0.05 based on Duncan’s post hoc test. Error bars represent means ± SD of three biological replicates.

Figure 2.

PpERF98-1 and 2 are transcriptional activators. A) Schematic diagrams of construct vectors used for transcriptional activity assays. The full-length and truncated fragments of PpERF98-1/2 were introduced downstream of the GAL4BD (galactose-specific transcription enhancing factor binding domain) in the pGBKT7 vector. PpERF98-1/2 ΔN and ΔC represent the deletion of the N and C termini of PpERF98-1/2 proteins, respectively. Numbers above shaded boxes indicate the position of nucleotides. AP2, APETALA2-domain; EDLL, a potent plant transcriptional activation domain. B) Growth of the yeast strain AH109 (Saccharomyces cerevisiae) transformed with the vectors, along with the negative control (pGBKT7), on synthetic dextrose (SD)/-Trp, SD/-Trp-His-Ade and SD/-Trp-His-Ade added with X-α-gal. C and D) Subcellular localization of PpERF98-1/2 based on the visualization of the GFP signal. The fusion construct PpERF98-1/2-GFP was co-transformed with the nuclear marker gene VirD2NLS fused to mCherry into Nicotiana benthamiana leaves. Confocal microscopic images of the cells were taken for GFP, mCherry fluorescences or bright field. Scaleb bar = 20 μm.

Figure 3.

VIGS of PpERF98-1 and 2 increases peach seedlings resistance to L. theobromae infection. A and B) Relative transcript levels of PpERF98-1/2 in PNRSV-mediated VIGS seedlings and the negative control (PNRSV). C) Phenotype of PpERF98-1/2-silenced peach leaves at 24 and 48 hpi. Bar = 1 cm. D and E) Lesion size and relative fungal biomass in the PpERF98-1/2-silenced peach leaves inoculated with L. theobromae.F to J) Relative transcript levels of PR genes (PRs; PpPR1, PpPR2, PpPR3, PpPR4, and PpCHI) in the PpERF98-1/2-silenced peach leaves infected with L. theobromae. Error bars represent means ± Sd of 3 independent replicates. Asterisks on top of bars indicate statistical significance with *P < 0.05 and **P < 0.01 using Student's t test, while ns indicates no significance.

Figure 4.

OE of PpERF98-1 and 2 in peach and tomato leaves attenuates resistance to L. theobromae infection. A and B) Relative transcript levels of PpERF98-1/2-overexpressing peach seedling lines (#1 and #2). Peach seedlings transformed with the empty vector and inoculated with L. theobromae were regarded as controls (empty). C) Phenotype of PpERF98-1/2-overexpressing peach leaves inoculated with L. theobromae at 24 and 48 hpi. Leaf images were digitally extracted with a same scale, and bar = 1 cm. D) Lesion areas in L. theobromae-infected leaves of the PpERF98-1/2-overexpressing peach seedlings. E to I) Relative transcript levels of PRs (PpPR1, PpPR2, PpPR3, PpPR4, and PpCHI) in the infected leaves of PpERF98-1/2-overexpressing seedlings. J and K) Leaf symptoms and lesion areas in tomato wild type (WT) and lines (#1 and #2) constitutively overexpressing PpERF98-1/2 and inoculated with L. theobromae. Leaf images were digitally extracted with a same scale, and bar = 1 cm. L and M) Relative transcripts levels of PRs (SlPR1, SlPR2, SlPR5, SlPRNP24, SlPR4, and SlCHI) in the PpERF98-1/2-overexpressing tomato lines infected with L. theobromae (Lt) or treated with a sterile PDA plug (Mock). Relative transcript levels were normalized on the reference genes PpTEF2 in peach and SlActin in tomato and are displayed as fold change compared with mock samples at 0 hpi (which were therefore set to 1). Error bars represent means ± Sd of 3 independent replicates. Asterisks on top of bars indicate statistical significance with *P < 0.05 and **P < 0.01 using Student's t test, whereas ns indicates no significance.

Figure 5.

PpERF1-1/2 negatively regulate peach defense against L. theobromae infection, and are activated by PpERF98-1/2. A and B) Time-course transcripts of PpERF1-1/2 in shoots infected with L. theobromae. C) Lesion phenotype of the infected peach leaves of PpERF1-1/2-overexpressing plants at 24 and 48 hours post inoculation (hpi). Peach seedlings transformed with the empty vector and inoculated with L. theobromae were regarded as controls (Empty). Leaf images were digitally extracted with a same scale, bars = 2 cm. D and E) Lesion size and relative fungal biomass in the infected leaves of peach seedlings transiently overexpressing PpERF1-1/2. Data are means ± SD of three independent replicates and four analytical replicates. F) Schematic diagram of different fragments (Fn) used for reporter constructs for luciferase (LUC) activity assay. The pentagram and rectangle stand for the putative ERF binding site and the GCC-box (GCCGCC), respectively, in the promoters of PpERF1-1/2 (proPpERF1-1/2). Numbers below the fragments indicate the positions of the nucleotides at the 5'-3' end of each fragment relative to the translation start-site in reporters. G and H) Transient expression assays showing that PpERF98-1/2 could activate the expression of PpERF1-1/2. Luminescence imaging of N. benthamiana leaves is shown 48 h after co-infiltration with different pairs of reporter and effector constructs. pGreenII-62-SK(62-SK)+pGreenII-0800-promoter-LUC(proPpERF1-1/2 Fn) was the control on the left, and 62-SK-PpERF98-1/2(PpERF98-1/2)+pGreenII-0800-promoter-LUC(proPpERF1-1/2 Fn) was the treatment on the right in one N. benthamiana leaf. I and J) Activation of proPpERF1-1/2 by PpERF98-1/2 in N. benthamiana leaves, as measured by the transient dual LUC assay. Data are means ± SD of five independent replicates. K) Schematic diagram of fragments (Pn) of proPpERF1-1/2 used as baits in yeast one-hybrid assay. L) Interaction analysis of PpERF98-1/2 with the fragments of proPpERF1-1/2. Transformed yeast cells containing both prey and bait were cultured on the selective medium SD/-Leu-Ura+AbA (aureobasidin A). Co-transformation of pGADT7-53 and pAbAi-p53 was used as positive and of pGADT7 and pAbAi-bait as negative controls, respectively. Asterisks on top of bars show statistical significance between transgenic and control plants at same time point and P < 0.05 using Student's t test, while ns indicates no significance.

Figure 6.

PpERF98-1 and 2 form heterodimers and homodimers and interact with PpERF1-1/2 in aggravating peach seedlings susceptibility to L. theobromae. A) In vivo interaction between PpERF98-1 and PpERF98-2 by BiFC assay in N. benthamiana leaves. Bar = 20 μm. B) Heterodimer formation between PpERF98-1/2 and PpERF1-1/2 in vivo by BiFC analysis. Bar = 20 μm. C) Schematic diagram of the construct combinations for LCI analysis, showing 1 experimental group (Gene1-nLUC + Gene2-cLUC) and 3 parallel controls in 1 tobacco leaf. D) Interactions between PpERF98-1 and PpERF98-2 by LCI. E and F) Interactions between PpERF98-1/2 and PpERF1-1/2 as determined by LCI. Combinations of nLUC or cLUC with the corresponding PpERF98-1/2 and PpERF1-1/2 constructs were used as negative controls. G and H) Lesion phenotype and size in peach leaves after coinfiltration with PpERF98-1/2-OE and PpERF1-1/2-OE constructs in Agrobacterium for 48 h and inoculation with L. theobromae at 24 and 48 hpi. Bar = 2 cm. Peach seedlings transformed with the empty vector and inoculated with L. theobromae were regarded as controls (empty). Leaf images were digitally extracted with a same scale in panel G), bar = 2 cm. Different letters show statistical significance of different treatments at the same time point and P < 0.05 based on Duncan's post hoc test. Error bars represent are means ± Sd of 3 independent replicates.

Figure 7.

The defense pathway of JA/ET and SA are regulated by PpERF98-1/2. A and B) Accumulation of JA and SA in the PpERF98-1/2-silenced peach seedlings at 0 and 24 hpi with L. theobromae. C to F) Lesion symptom and size in the hormone-treated leaves of the susceptible cultivar SS and the tolerant cultivar DHP prior to L. theobromae inoculation. Fully expanded leaves were evenly sprayed with 100 µM MeJA, 500 µM SA, 500 µM ACC (ET precursor, 1-aminocyclopropane-1-carboxylate), or ddH₂O for 24 h prior to L. theobromae or mock inoculation. Leaf images were digitally extracted with a same scale in panes C) and D), bar = 2 cm. Different letters on top of bars indicate statistical significance at the same time point and P < 0.05 based on Duncan's post hoc test. G) Proposed regulatory model for the role of PpERF98-1 and 2 in peach response to the gummosis fungus L. theobromae. When plants are infected by L. theobromae, large amounts of ET are produced and induce the transcription of PpERF98-1/2. These 2 PpERF98 proteins form heterodimers and homodimers with themselves and directly bind to the GCC-box cis-elements of the promoters of PpERF1-1/2, activating their transcription. Further, these 2 PpERF98 proteins interact with 2 PpERF1 proteins to form heterodimers and amplify JA/ET signaling. Moreover, the PpERF98s can also activate the transcription of the JA/ET-responsive genes PpPR3, PpPR4, and PpCHI through putative ERF-binding cis-elements. Blocking the JA and ET pathways can decrease peach susceptibility to L. theobromae, while SA can promote tolerance (our earlier work; Zhang et al. 2022). Altogether, our data show that 2 interacting PpERF98 genes negatively regulate peach resistance to L. theobromae by activating JA/ET signaling to attenuate the SA-dependent defense pathway. Red bars represent GCC-box, and solid green circles indicate other putative ERF-binding sites in target genes.