<i>Phytophthora sojae</i> Effector PsCRN70 Suppresses Plant Defenses in <i>Nicotiana benthamiana</i>

<div><p><i>Phytophthora sojae</i>, an oomycete pathogen, produces a large number of effector proteins that enter into host cells. The Crinklers (Crinkling and Necrosis, CRN) are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infective stages in <i>P. sojae</i>. However, their roles in pathogenesis are largely unknown. Here, we functionally characterized an effector <i>PsCRN70</i> by transiently and stably overexpressing it in <i>Nicotiana benthamiana</i>. We demonstrated that PsCRN70 was localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the <i>PsCRN70</i> gene in <i>N. benthamiana</i> enhanced susceptibility to <i>P. parasitica</i>. The H₂O₂ accumulation in the <i>PsCRN70</i>-transgenic plants was reduced compared to the <i>GFP</i>-lines. The transcriptional levels of the defense-associated genes, including <i>PR1b</i>, <i>PR2b</i>, <i>ERF1</i> and <i>LOX</i>, were also down-regulated in the <i>PsCRN70</i>-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H₂O₂ accumulation and the expression of defense-associated genes, and therefore promotes pathogen infection.</p></div

Suppression of the H₂O₂ accumulation in <i>N. benthamiana</i> by PsCRN70.

<p>A. DAB staining of the <i>P. parasitica</i>-inoculated <i>N. benthamiana</i> leaves. The H₂O₂ accumulation in the <i>PsCRN70</i>- and <i>GFP</i>- transgenic leaves were detected using DAB staining at 12 hpi. Photographs were taken after de-colorization of leaves with ethanol. B. The relative levels of DAB staining. The data were calculated by a combination of Photoshop and Quantity One for H₂O₂ accumulation in the indicated transgenic lines. The experiments were repeated three times in all transgenic lines with similar results. Four leaves were used for each treatment in each experiment. Bars represent the standard deviation (SD). Different letters at the top of the columns indicate significant differences (P<0.01, Duncan's multiple range test).</p

Down-regulation of the defense-associated genes in <i>N. benthamiana</i> by PsCRN70.

<p>A. Relative expression levels of the <i>PR1b</i>, <i>PR2b</i>, <i>ERF1</i> and <i>LOX</i> genes. The total RNA was extracted from the leaf tissues that transiently expressing <i>PsCRN70</i> and <i>GFP</i>, respectively, and the expression levels of the indicated genes were measured using qRT-PCR. B. Relative expression levels of the indicated genes in the stable transgenic <i>N. benthamiana</i>. The <i>N. benthamiana</i> leaves 36 hpi with <i>P. parasitica</i> zoospores were collected, and the gene expression levels were measured by qRT-PCR. The gene expression levels were normalized to the <i>EF1α</i> gene. Bars represent the standard deviation (SD), with significant difference (** for P<0.01 and * for P<0.05, Student's <i>t</i>-test).</p

Characterizations of the <i>PsCRN70-</i>transgenic <i>N. benthamiana</i>.

<p>A. RT-PCR analysis of <i>PsCRN70</i> expression in independent transgenic lines. #1, #3, #4 and #12, four independent T1 transgenic lines; P, <i>pBinGFP:PsCRN70</i> plasmid as a positive control; U, untransformed plant as a negative control. The upper panel represents the 624 bp fragment of <i>PsCRN70</i> gene and the lower panel represents the 100 bp fragment of <i>EF1α</i> gene as the control. B. Western blot analysis of expression of GFP: PsCRN70 fusion protein in transgenic <i>N. benthamiana</i> using monoclonal antibody against GFP. Subcellular localization of PsCRN70 in the transgenic <i>N. benthamiana</i> leaf tissues (C) and roots (D). The pictures were taken using a confocal microscope. The scale bar indicates 50 µm.</p

A Virulence Essential CRN Effector of <i>Phytophthora capsici</i> Suppresses Host Defense and Induces Cell Death in Plant Nucleus

<div><p><i>Phytophthora capsici</i> is a soil-borne plant pathogen with a wide range of hosts. The pathogen secretes a large array of effectors during infection of host plants, including Crinkler (CRN) effectors. However, it remains largely unknown on the roles of these effectors in virulence especially in <i>P</i>. <i>capsici</i>. In this study, we identified a cell death-inducing CRN effector PcCRN4 using agroinfiltration approach. Transient expression of <i>PcCRN4</i> gene induced cell death in <i>N</i>. <i>benthamiana</i>, <i>N</i>. <i>tabacum</i> and <i>Solanum lycopersicum</i>. Overexpression of the gene in <i>N</i>. <i>benthamiana</i> enhanced susceptibility to <i>P</i>. <i>capsici</i>. Subcellular localization results showed that PcCRN4 localized to the plant nucleus, and the localization was required for both of its cell death-inducing activity and virulent function. Silencing <i>PcCRN4</i> gene in <i>P</i>. <i>capsici</i> significantly reduced pathogen virulence. The expression of the pathogenesis-related gene <i>PR1b</i> in <i>N</i>. <i>benthamiana</i> was significantly induced when plants were inoculated with <i>PcCRN4</i>-silenced <i>P</i>. <i>capsici</i> transformant compared to the wilt-type. Callose deposits were also abundant at sites inoculated with <i>PcCRN4</i>-silenced transformant, indicating that silencing of <i>PcCRN4</i> in <i>P</i>. <i>capsici</i> reduced the ability of the pathogen to suppress plant defenses. Transcriptions of cell death-related genes were affected when <i>PcCRN4</i>-silenced line were inoculated on <i>Arabidopsis thaliana</i>, suggesting that PcCRN4 may induce cell death by manipulating cell death-related genes. Overall, our results demonstrate that PcCRN4 is a virulence essential effector and it needs target to the plant nucleus to suppress plant immune responses.</p></div

Expression of <i>PcCRN4</i> in <i>N</i>. <i>benthamiana</i> increased susceptibility to <i>Phytophthora</i> and suppressed ROS accumulation.

<p>(A) Observed phenotypes and (B) lesion diameters of <i>N</i>. <i>benthamiana</i> leaves inoculated with <i>P</i>. <i>capsici</i>. Ten μl (100 μl^-1) zoospores were inoculated in the infiltrated regions 24 hours post infiltrated with the indicated genes and the photograph was then taken at 36 hours post inoculation. The lesion diameters were scored at the indicated time points. Statistical analyses were performed using a Dunnett’s test. (**, P < 0.01). (C) DAB staining of the inoculated sites of <i>N</i>. <i>benthamiana</i>.</p

PcCRN4 effector domains function in plant nucleus.

<p>(A) Nuclear localization is required for PcCRN4-inducing cell death. <i>N</i>. <i>benthamiana</i> leaves were infiltrated with <i>Agrobacterium</i> strains carrying the indicated constructs. The representative pictures were taken at 5 dpi. The number shows the cell death sites and the total infiltrated leaves for each gene. NES and nes represent the nuclear export signal and nonfunctional NES. (B) NES impairs accumulation of GFP:PcCRN4 in <i>N</i>. <i>benthamiana</i> nucleus. <i>N</i>. <i>benthamiana</i> leaves were agroinfiltrated with the indicated constructs 48 hours before assessment of GFP confocal imaging. Scale bars, 25 μm. (C) Immunoblot analyses of GFP fusion protein accumulation <i>in planta</i>. Total proteins were extracted at 48 hpi. Blots were probed with α-GFP antibody. Sizes in kDa are indicated on the left.</p

<i>PcCRN4</i>-silenced line showed reduced virulence.

<p>(A) Generation of a transformant with the silenced <i>PcCRN4</i> gene. Relative expression of <i>PcCRN4</i> in the silenced line (T4), unsilenced line (CK) and the wild type (WT) is shown. <i>P</i>. <i>capsici actin</i> transcripts were used as a reference and then normalized to the wild-type. Each bar represents the mean of three independent experiments with SE. (B and C) Lesions induced by the silenced line T4 and WT on <i>N</i>. <i>benthamiana</i> (B) and <i>A</i>. <i>thaliana</i> (C) leaves. The typical photos were taken at 36 hpi (B) and 30 hpi (C). (D) Lesion diameters of the inoculated sites. The data was measured at 36 hpi (<i>N</i>. <i>benthamiana</i>) and 30 hpi (<i>A</i>. <i>thaliana)</i> from over three independent replicates (**, P < 0.01, Student <i>t</i>-test). (E) Relative DNA amount in <i>P</i>. <i>capsici</i> in <i>N</i>. <i>benthamiana</i>. The relative virulence was calculated by Q-PCR assays of pathogen DNA levels in infected leaves relative to host DNA at12 hpi. Error bars represent SD from three technical replicates. (**, P < 0.01, Student <i>t</i>-test). (F) Relative expression of <i>PR1b</i> in <i>N</i>. <i>benthamiana</i> leaves. <i>P</i>. <i>capsici actin</i> gene was used as a reference and then normalized to the uninfected leaves (0 h) (**, P < 0.01, Dunnett’s test).</p

Silenced line could not suppress host H₂O₂ accumulation and callose deposition.

<p>(A) Trypan blue staining of the inoculated <i>A</i>. <i>thaliana</i> leaves. The typical photographs were taken after decolorizing with chloral hydrate 24 hpi. Bar, 40 μm. (B). Reactive oxygen species (ROS) accumulation. The inoculated leaves were stained with DAB stain and photographs were taken at 12 hpi. (C) Callose deposition detected with aniline blue staining. Data under the photos shows the relative callose intensities at 6 and 24 hpi from four replicates. The average number of callose deposits per microscopic field of 1 mm² was calculated using the ImageJ software. Bars, 100 μm. T4, silenced line, WT, wild-type.</p

Relative expression levels of cell death-related genes in <i>Arabidopsis</i> after infection with wild-type and <i>PcCRN4</i>-silenced line.

<p>The relative levels of transcript were calculated by the comparative Ct method. Expression on leaves of <i>A</i>. <i>thaliana</i> inoculated with pathogen for 0 hr was fixed as one. Transcript levels of <i>UBQ5</i> gene of <i>Arabidopsis</i> were used to normalize different samples. Bars represent means and standard deviations of three replications. Asterisks indicate statistical differences between the transcripts of WT and T4 (P<0.01, Dunnett’s test).</p