A barley powdery mildew fungus non-autonomous retrotransposon encodes a peptide that supports penetration success on barley

Abstract

Pathogens overcome plant immunity by means of secreted effectors. Host effector targets often act in pathogen defense, but might also support fungal accommodation or nutrition. The barley ROP GTPase HvRACB is involved in accommodation of fungal haustoria of the powdery mildew fungus Blumeria graminis f.sp. hordei (Bgh) in barley epidermal cells. We found that HvRACB interacts with the ROP-interactive peptide 1 (ROPIP1) that is encoded on the active non-long terminal repeat retroelement Eg-R1 of Bgh. Overexpression of ROPIP1 in barley epidermal cells and host-induced post-transcriptional gene silencing (HIGS) of ROPIP1 suggested that ROPIP1 is involved in virulence of Bgh. Bimolecular fluorescence complementation and co-localization supported that ROPIP1 can interact with activated HvRACB in planta. We show that ROPIP1 is expressed by Bgh on barley and translocated into the cytoplasm of infected barley cells. ROPIP1 is recruited to microtubules upon co-expression of MICROTUBULE ASSOCIATED ROP GTPase ACTIVATING PROTEIN (HvMAGAP1) and can destabilize cortical microtubules. The data suggest that Bgh ROPIP targets HvRACB and manipulates host cell microtubule organization for facilitated host cell entry. This points to a possible neo-functionalization of retroelement-derived transcripts for the evolution of a pathogen virulence effector.

Fig. 1.

Bgh ROPIP1 and ROPIP1-Cter interacted with barley HvRACB and CA HvRACB in yeast. (A) ROPIP1 of Bgh was tested as prey in targeted Y2H assays for interaction with the barley small GTPase HvRACB in three different variants: WT, wild-type protein; CA, constitutively activated mutant (HvRACB G15V); DN, dominant negative mutant (HvRACB T20N) and with the HvRACB-interacting protein HvMAGAP1. The ROPIP1 sequence was additionally split into its small inherent C-terminal ORF (ROPIP1-Cter) which was sufficient for protein interaction with WT HvRACB and CA HvRACB and the remaining N-terminal part (ROPIP1-Nter) which did not interact with the baits. A total of 10⁵ cells of each combination were dropped in parallel on SD-Leu,-Trp (-L-W) as transformation control and on SD-Ade,-His,-Leu,-Trp (-A-H-L-W) selection medium. (B) Serial dilution of 10⁵–10 yeast cells transformed with pGADT7-ROPIP1 as prey vector and pGBKT7-HvRACB WT as bait vector or pGBKT7-empty as empty vector control. Left panel: transformation control medium (SD-L-W). Right panel: selection medium (SD-A-H-L-W) supplemented with 2.5 mM 3-AT to increase selectivity. (This figure is available in colour at JXB online.)

Fig. 2.

ROPIP1 modulated susceptibility of barley epidermal cells towards Bgh. (A) Transient overexpression of ROPIP1 and ROPIP1-Cter in barley epidermal cells significantly increased the relative penetration rate of Bgh in comparison with the control. (B) Host-induced gene silencing (HIGS) of native ROPIP1 by transient expression of ROPIP1 as dsRNA (ROPIP1-RNAi) in barley epidermal cells significantly decreased the relative penetration rate of Bgh. Co-expression of a ROPIP1-RNAi-rescue construct (RNAi rescue) significantly complemented HIGS of the native ROPIP1 transcript. Bars represent the mean values of six independent experiments in (A) and four independent experiments in (B). Error bars are ±SE. *P≤0.05 (Student’s t-test).

Fig. 3.

Western blot of barley leaf protein extracts using α-ROPIP1 antibody. (A) Affinity-purified anti-peptide antibody α-ROPIP1 was used as the primary antibody in western blots of total protein extracts prepared from barley leaves inoculated (+Bgh) or non-inoculated (–Bgh) with Bgh. His-tag purified recombinant ROPIP1 (recROPIP1) was run as a positive control on the same gel. RecROPIP1 and a protein exclusive to the +Bgh sample were labeled by α-ROPIP1. Several repetitions confirmed the signal in the +Bgh lane. (B) Controls for α-ROPIP1 specificity. Escherichia coli Rosetta cells were transformed with the IPTG-inducible vector pET28b:ROPIP1. Crude cell lysates were prepared from small-scale cell cultures with (+) or without (–) IPTG induction. Recombinant His-tagged ROPIP1 was detected by α-ROPIP1 in the IPTG-induced sample (+) but not in the non-induced control (–). The use of α-His antibody in aliquots of the same samples validated the identity of the signal. The experiment was repeated twice with identical results. Ponceau S: loading and protein transfer control. The arrowhead points to a faint band in the recROPIP1 lane in (A). MW, molecular weight protein ladder; PE, protein extract. (This figure is available in colour at JXB online.)

Fig. 4.

Immunogold labeling of α-ROPIP1 in Bgh-challenged barley leaves. Transmission electron micrographs of ultrathin sections of Bgh-infected barley epidermal cells 3 dai showing gold particles bound to α-ROPIP1. (A, B) Negative control of infected cells treated with a non-specific antibody. Gold particles were absent in the susceptible barley epidermal cell containing intracellular fungal haustorial protrusions (H) and the extracellular Bgh hypha (Hy). (C, D) Gold particles bound to α-ROPIP1 were observed in hyphae, inside a Bgh appressorium (App), the barley epidermal cell wall (CW), and papilla, but were absent from the extracellular space (ES) and the host cell vacuole (V). (E, F) Gold particles were found in the lumen of finger-like Bgh haustorial protrusions inside barley epidermal cells as well as the host cell cytoplasm, but were almost absent from the host cell vacuole (V), the CW, and the ES. Arrowheads in (D) and (F) point to selected gold particles. Scale bars are 1 µm.

Fig. 5.

Recruitment of GFP–ROPIP1 to cortical microtubules (MTs) by RFP–HvMAGAP1. Barley leaf epidermal cells were transiently transformed by particle bombardment and imaged with confocal laser scanning microscopy as sequential whole-cell scans in 2 µm increments at 12–24 hat. (A) Whole-cell projection showing cytoplasmic and unspecific subcellular localization of GFP–ROPIP1. Co-localization with cytoplasmic and nucleoplasmic mCherry fluorescence is indicated by white pixels in the merge picture. The observation was consistently repeatable in more than three experiments. (B) Recruitment of GFP–ROPIP1 to cortical MTs upon co-expression of MT-associated RFP–HvMAGAP1. White pixels in the merge picture indicate co-localization. A maximum projection of 20 optical sections in 2 µm increments is shown. The observation was consistently repeatable in more than three experiments. (C) Visualization of co-expressed fusion protein combinations used for quantitative analysis. C-ter, truncation of HvMAGAP1 to the MT-associated C-terminus (HvMAGAP1-Cter); FL, full-length HvMAGAP1. Ten optical sections of the upper cell cortex were merged for the pictures. (D) Quantification of the combinations shown in (C). Bars are frequencies of cells with GFP fluorescence being located at MTs or in the cytoplasm only (CYT) derived from three independent experiments. The respective absolute numbers of the categories were compared in a χ² test. RFP–HvMAGAP1-Cter highly significantly reduced MT association of GFP–ROPIP1 (***P≤0.001, n=61, 60, 53, and 57 cells from left to right). Scale bars in (A), (B), and (C) are 20 µm.

Fig. 6.

Split YFP complementation of ROPIP1–YFP^N and YFP^C–CA HvRACB in planta. (A) ROPIP1–YFP^N was transiently co-expressed with DN or CA (right) YFP^C–HvRACB, the inactive RFP–HvMAGAP1-R185G mutant, and CFP as a transformation marker in barley leaf epidermal cells. Confocal laser scanning microscopy whole-cell maximum projections are shown. (B) Detailed picture of the ROPIP1–YFP^N and YFP^C–CA HvRACB co-expressing cell from (A) (dashed square). A maximum projection of 10 optical sections at 2 µm from the upper cell cortex is shown. Scale bars in (A) and (B) are 20 µm. (C) Ratiometric measurement of YFP fluorescence complementation. ROPIP1–YFP^N was transiently co-expressed with YFP^C–CA HvRACB or YFP^C– DN HvRACB, and YFP signals were normalized to signals from co-expressed CFP. Error bars are ±SE. Two-sided Student’s t-test (**P≤0.01). (D) Co-expression of GFP–ROPIP1, CFP–CA HvRACB, and RFP–HvMAGAP1. Transformed cells were imaged as whole-cell scans by confocal laser scanning microscopy at 48 hat. GFP–ROPIP1, CFP–CA HvRACB, and RFP–HvMAGAP1 showed similar localization at the cell periphery and at microtubules. The scale bar is 20 µm.

Fig. 7.

Co-expression of GFP–ROPIP1 and RFP–HvMAGAP1 enhanced microtubule (MT) network disorganization. (A) Example micrographs illustrating three distinct categories of MT network organization in barley epidermal cells. Confocal laser scanning microscopy whole-cell projections of barley epidermal cells transiently co-expressing GFP–ROPIP1 and RFP–HvMAGAP1 are shown in gray scale. Scale bars are 20 µm. (B) Mean relative frequencies of the categories at 12–24 hat. The absolute numbers of cells per category of n=145 GFP and n=132 GFP–ROPIP1 cells each co-transformed with RFP–HvMAGAP1 obtained from four independent repetitions were compared in a χ² test (***P≤0.001; χ²=27.92; df=2). Cells of category 3 exhibiting a heavily disorganized MT network tripled from 15.5% in the GFP control to 44.3% in cells expressing GFP–ROPIP1.