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. 2025 Jul 18:16:1570072.
doi: 10.3389/fmicb.2025.1570072. eCollection 2025.

Effector Pt 31812 from Puccinia triticina acts as avirulence factor for Lr42-mediated resistance in wheat

Jianyuan Li  1   2 , Jiali Li  3 , Jie Wei  1   4 , Lin Li  2 , Yue Qi  1   5 , Yue Zhang  1   6 , Wenxiang Yang  1 , Qian-Hua Shen  3
Affiliations

Effector Pt 31812 from Puccinia triticina acts as avirulence factor for Lr42-mediated resistance in wheat

Jianyuan Li et al. Front Microbiol. .

Abstract

As an obligate biotrophic fungus, the leaf rust pathogen Puccinia triticina (Pt) secretes a repertoire of effector proteins into host cells for modulating plant immunity and promoting fungal pathogenesis. Here, we identify the Pt31812 effector and characterize its function in pathogenesis and immune-related activity in plants. In the study, Pt31812 was cloned by PCR, and the expression pattern and structure were analyzed by qRT-PCR and online softwares. Subcellular localization of Pt31812 was analyzed using transient expression on Nicotiana Benthamiana. Further functional analysis was conducted using transient expression and host-induced gene silencing (HIGS). The results showed that Pt31812 encodes candidate effector with a predicted signaling peptide (SP) at the N-terminus, and its expression was highly up-regulated during Pt infection of wheat. Subcellular localization analysis revealed that Pt31812 is localized in cytoplasm and nucleus when expressed in N. Benthamiana. Co-expression of Pt31812 and mammalian BAX protein revealed that Pt31812 inhibited BAX-induced cell death in N. Benthamiana, and the fragment of 22-88 aa from the N-terminus of the effector was important for the inhibiting activity. Interestingly, expression of Pt31812 in a panel of wheat differential lines with different Lr resistance genes showed that Pt31812 specifically triggered cell death in a Lr42-harboring wheat line. Furthermore, transient gene silencing of Pt31812 through BSMV-HIGS approach rendered loss of Lr42-mediated resistance against rust race Pt.-THSN and altered the infection type from resistant to susceptible. Our data reveal that Pt31812, as a candidate effector with immune inhibiting activity, acts as an avirulence determinant factor during Pt infection of Lr42-harboring wheat line. These findings highlight immune-related activity of specific Pt effectors and lay the foundation for further investigation into mechanisms of leaf rust fungal pathogenesis and recognition.

Keywords: Lr42; Pt31812; avirulence gene; effector; pathogenesis; wheat leaf rust.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Bar graph showing relative expression of Pt31812 at various hours post-infection (hpi): 0, 6, 12, 18, 24, 36, 48, 60, 72, 96, and 120. Significant increases in expression are marked with asterisks, peaking at 96 and 120 hpi.
FIGURE 1
Pt31812 expression is highly up-regulated during Pt. infection of wheat. Pt31812 transcript level were analyzed by qRT-PCR analysis during Pt. infection of leaves of wheat cv. Thatcher from 0 to 120 hpi. Relative expression was calculated by the comparative Ct method with EF1 as a reference gene. Error bars = ± SD (n = 3 for biological replicates). ** for P < 0.01; Statistic analysis was performed by Student’s t-test.
Fluorescent microscopy images showing leaf epidermal cells. The top row shows GFP expression with a bright field and merged images. The bottom row shows the P31812(?SP)-GFP variant with corresponding bright field and merged images. Green fluorescence highlights cellular structures against a grayscale background.
FIGURE 2
Pt31812 is localized in cytoplasm and nucleus in N. benthamiana. GFP and Pt31812(△SP)-GFP fusion proteins were transiently expressed in N. benthamiana via Agrobacterium-infiltration. Confocal imaging was done at 48 hpi. Scale bar = 5 μm.
Diagram A shows a circle divided into four sections labeled GFP, GFP + BAX, Effector-GFP, and Effector-GFP + BAX. Images B and C show leaves each with six marked circles, indicating areas of treatment or observation. The leaves in image B display darker regions within the circles compared to image C.
FIGURE 3
Pt31812 suppresses BAX-induced cell death in N. benthamiana. (A) Scheme of Agrobacterium-infiltration. (B,C) Expression of GFP or effector-GFP fusion alone (left half), or coexpression with BAX (right half) in leaves of N. benthamiana, and Effector-GFP fusion represents Pt31812(FL)-GFP (B) or Pt31812(△SP)-GFP (C). The cell death phenotype was photographed at 5 d post infiltration.
Diagram consisting of three parts. Part A shows a schematic of protein sequences labeled Pt31812 with various lengths indicated (209 aa, 178 aa, 148 aa, 118 aa, 88 aa). Part B displays small images of leaves corresponding to each protein sequence. Part C features a leaf with six marked sections, each linked to a different protein-BAX combination, as explained in the adjacent legend.
FIGURE 4
The region of 22–88 aa in Pt31812 is important for suppressing BAX-induced cell death. (A) Schematic representation of Pt31812 deletion variants. (B) expression of Pt31812 deletion variants did not induce cell death upon transient expression in N. benthamiana via Agrobacterium-infiltration. (C) Co-expression of Pt31812 deletion variants with BAX in N. benthamiana leaves, with GFP as a control. Images were taken at 5 dpi.
Close-up of seven wheat stems labeled with numbers one to six and “EV” under the heading “Pt31812.” The stems, belonging to the Lr42-harboring wheat line, display varying levels of discoloration and dark markings.
FIGURE 5
Expression of Pt31812 induces cell death in a Lr42-harboring wheat line. Transient expression of Pt31812 or empty vector (EV) in leave of Lr42-harboring wheat via Agrobacterium infiltration. Representative images were photographed 7 dpi. The two black lines mark the Agrobacterium-infiltrated region in the leaf.
Graph and images illustrate the effect of BSMV:Pt31812 on wheat. Part A shows a bar graph comparing relative expression levels at 24, 48, and 120 hours post-inoculation, with BSMV:Pt31812 showing consistently lower expression than BSMV:00. Part B displays images of wheat leaves from the Lr42 line under different treatments: CK, BSMV:00, BSMV:Pt31812, and BSMV:PDS, highlighting differences in leaf appearance.
FIGURE 6
Silencing of Pt31812 converts avirulent Pt.-THSN to a virulent isolate in Lr42-harboring wheat line. (A) Transcript levels of Pt31812 were determined by qRT-PCR at 24, 48, and 120 hpi of Pt. isolate in BSMV:00 or BSMV:Pt31812 treated leaves of a Lr42-harboring wheat line. * for P < 0.05. (B) Disease phenotypes of Pt.-THSN infected leaves upon BSMV treatment. The second leaves of Lr42-harboring wheat line were treated by sodium phosphate buffer (CK), or BSMV-HIGS vector (BSMV:Pt31812), or empty vector control (BSMV:00). Images were taken at 10 dpi.
Four microscopic images of Lr42-harboring wheat line show cellular structures at 24 and 48 hours post-inoculation (hpi). Panel A (24 hpi, BSMV: 00) and Panel C (48 hpi, BSMV: 00) display structures labeled AP, SV, IH, HMC, NC, and H. Panel B (24 hpi, BSMV: Pt31812) and Panel D (48 hpi, BSMV: Pt31812) show structures labeled AP, SV, IH, HMC, H, and SH. Blue and green fluorescence highlights different cellular features. Each image is marked with scale bars.
FIGURE 7
Silencing of Pt31812 results in loss of Lr42-mediated cell death in wheat. (A–D): Confocal imaging of Pt.-THSN infected leaf cells of Lr42-harboring wheat line at 24 hpi and 48 hpi. Wheat leaves were treated with BSMV empty vector (BSMV:00) or silencing vector (BSMV: Pt31812) followed by inoculation of Pt.-THSN uredospores. AP: appresorium; IH: infection hypha; SV: substomatal vesicle; HMC: haustorial mother cell; SH: second hypha; H: haustorium; NC: necrotic cell. Scale bar = 30 μm.
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