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Proc Natl Acad Sci U S A. Dec 20, 2011; 108(51): 20832–20837.
Published online Dec 5, 2011. doi:  10.1073/pnas.1112708109
PMCID: PMC3251060
Plant Biology

Phytophthora infestans effector AVRblb2 prevents secretion of a plant immune protease at the haustorial interface

Abstract

In response to pathogen attack, plant cells secrete antimicrobial molecules at the site of infection. However, how plant pathogens interfere with defense-related focal secretion remains poorly known. Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen Phytophthora infestans focally accumulates around haustoria, specialized infection structures that form inside plant cells, and promotes virulence by interfering with the execution of host defenses. AVRblb2 significantly enhances susceptibility of host plants to P. infestans by targeting the host papain-like cysteine protease C14 and specifically preventing its secretion into the apoplast. Plants altered in C14 expression were significantly affected in susceptibility to P. infestans in a manner consistent with a positive role of C14 in plant immunity. Our findings point to a unique counterdefense strategy that plant pathogens use to neutralize secreted host defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.

Keywords: plant cell-autonomous immunity, polarized secretion, late blight

To enable parasitism and symbiosis, plant-associated organisms intimately interact with plant cells often through specialized cellular structures. Some biotrophic fungal and oomycete pathogens form accommodation structures termed haustoria that invaginate the host cell plasma membrane to deliver pathogenicity effector proteins and acquire nutrients (1, 2). In response to and to restrict pathogen colonization, the attacked plant cell undergoes significant cellular reorganization, involving organelle relocation, cell-wall reinforcements around contact sites, and polarized secretion of antimicrobial molecules (3, 4).

An important group of host-secreted defense components are papain-like cysteine proteases (PLCPs). As a countermeasure, effective pathogens such as Phytophthora infestans, the oomycete pathogen that causes potato late blight, secrete extracellular protease inhibitors of cysteine proteases (EPICs) that bind and inhibit PLCPs in the apoplast (5). The existence of protease inhibitors from unrelated pathogens, such as Cladosporium fulvum Avr2 and P. infestans cystatin-like EPIC2B, that both target and inhibit apoplastic PLCPs RCR3 and PIP1 of tomato points to a key role of this group of proteases in immunity (6). Furthermore, a secreted PLCP RD19 from Arabidopsis is targeted and mislocalized to the host cell nucleus by the bacterial type III secreted effector PopP2 from Ralstonia solanacearum (7). Given the importance of apoplastic host defenses, it is likely that P. infestans has established multiple strategies to counteract secreted defense components, potentially including direct targeting of elements of the polarized secretion pathway.

The P. infestans genome encodes large families of host-translocated effectors (8, 9). The best-studied group of P. infestans effectors is the RXLR effector family, named for the presence of a conserved arginine-X-leucine-arginine motif. RXLR effectors operate inside the host cell to enable successful infection. Similar to other RXLR effectors, AVRblb2 (PexRD40170–7) (10) is a modular protein with the N-terminal half comprising a signal peptide and the RXLR domain involved in trafficking to host cell cytoplasm and the C-terminal region carrying the biochemical effector activities (10). As noted for other RXLR effectors with avirulence activity, Avrblb2 and its paralogs are sharply up-regulated during infection, peaking early during biotrophy (8, 10). These genes are important for P. infestans fitness because every known strain of the pathogen carries multiple intact coding sequences (10). Members of the AVRblb2 family are recognized inside plant cells by the broad-spectrum resistance protein Rpi-blb2 of the wild potato Solanum bulbocastanum (10). However, the primary activity of AVRblb2 and other RXLR effectors is to promote virulence, and the precise modes of action and host targets of these effectors remain largely unknown (11). Only recently, the RXLR effector AVR3a was shown to manipulate plant immunity by stabilizing the host E3 ligase CMPG1 (12). However, the extent to which plant pathogen effectors interfere with defense-related focal secretion is poorly known.

Here we show that the host-translocated RXLR-type effector protein AVRblb2 of the Irish potato famine pathogen P. infestans focally accumulates around haustoria inside plant cells and promotes virulence by interfering with the execution of polarized host defenses. Furthermore, we demonstrate that AVRblb2 targets PLCP C14 and prevents its secretion into the apoplast. C14 knockdown via RNAi-mediated silencing results in enhanced susceptibility toward P. infestans and promotes its hyphal growth. We present evidence that C14 is a unique plant defense protease and its overexpression limits P. infestans infection efficiency. However, this effect is partially reversed by in planta overexpression of AVRblb2. Our data point to a unique counterdefense strategy that plant pathogens use to neutralize secreted plant defense proteases. Effectors, such as AVRblb2, can be used as molecular probes to dissect focal immune responses at pathogen penetration sites.

Results and Discussion

AVRblb2 Localizes to the Cell Periphery and Accumulates Around Haustoria in Infected Cells.

To gain insight into AVRblb2 virulence activities inside host cells, we constructed a functional N-terminal GFP fusion to mature AVRblb2 (lacking the signal peptide) (Fig. S1 A and B). The GFP:AVRblb2 fusion protein accumulated mainly at the cell periphery when expressed in Nicotiana benthamiana (Fig. 1A). The fluorescence signal remained associated with the plasma membrane after salt-induced plasmolysis of the epidermal cells (Fig. 1A). The AVRblb2 signal largely overlapped with the red fluorescence of a coexpressed plasma membrane-localized RFP (pm-RK), confirming that AVRblb2 accumulates at the host plasma membrane (Fig. 1A). Similar membrane localization of AVRblb2 was observed with an N-terminal RFP fusion (Fig. 1A) and in stable transgenic GFP:AVRblb2 lines of N. benthamiana (Fig. S1C). To determine the extent to which localization of AVRblb2 is altered during infection, we inoculated the GFP:AVRblb2 N. benthamiana lines with several P. infestans strains, including 88069td, a transgenic strain expressing the red fluorescent marker tandem dimer RFP (known as tdTomato) (13). The AVRblb2 signal preferentially accumulated around haustoria inside infected plant cells, whereas its even distribution at the plasma membrane remained unaltered in cells without haustoria (Fig. 1B). Haustorial accumulation of AVRblb2 first occurred at discrete focal sites (one per haustorium) before covering the entire surface of the haustoria (Fig. 1B and Fig. S1C). Perihaustorial accumulation of AVRblb2 only partially overlapped with callose deposited around haustoria and was not associated with callose encasements (Fig. S1D) (14).

Fig. 1.
AVRblb2 localizes to the plasma membrane and accumulates around haustoria to promote P. infestans virulence. (A) Agrobacterium tumefaciens-mediated transient expression of GFP:AVRblb2 or RFP:AVRblb2 fusion proteins revealed peripheral localization, which ...

AVRblb2 Enhances P. infestans Virulence.

To determine the degree to which AVRblb2 affects P. infestans infection, we performed pathogen assays with the transgenic GFP:AVRblb2 N. benthamiana plants. The GFP:AVRblb2 plants showed enhanced susceptibility to P. infestans, resulting in increased pathogen colonization and sporulation relative to control lines (Fig. 1 C and D and Fig. S2). This finding indicates that AVRblb2 has a virulence activity and suggests that it causes significant impairment of host defense responses.

AVRblb2 Associates with C14 Protease in Planta.

To identify the host targets of AVRblb2, we used in planta coimmunoprecipitation (co-IP) followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS). In total, we detected five N. benthamiana proteins that specifically associated with AVRblb2 (Table S1). One of these targets is the PLCP C14, a conserved solanaceous protein orthologous to Arabidopsis RD21, rice Oryzain, and maize Mir3 cysteine proteases (1520). C14 is a complex modular protein featuring a predicted N-terminal secretion signal and a self-inhibitory prodomain, which is followed by peptidase, proline-rich, and granulin domains (Fig. 2A). In plant cells, C14/RD21 converts into immature (iC14) and mature (mC14) isoforms (Fig. 2A) that accumulate into various subcellular compartments and the apoplast (1618). C14 and other PLCPs have been implicated in plant immunity, including pathogen perception and disease resistance (5, 21). Like some other PLCPs, C14 is also targeted by apoplastic protease inhibitor effectors of P. infestans and other filamentous pathogens (5, 6, 18), and the expression of the potato C14 gene is rapidly induced during P. infestans infection (15). Thus, we decided to initially focus on C14, and we will study other AVRblb2-associated proteins in the future.

Fig. 2.
AVRblb2 associates with C14 in planta. (A) Domain organization of C14. C14 accumulates in cells as immature (iC14) and mature (mC14) isoforms. (B) AVRblb2 coimmunoprecipitates with C14 in planta. FLAG:Avrblb2 or FLAG:Avr3a was transiently coexpressed ...

We validated the specific in planta association between AVRblb2 and tomato C14 (LeC14) with co-IP (Fig. 2B). AVRblb2-purified immunocomplexes contained iC14 (Fig. 2B), and, occasionally, the less abundant mC14 isoform of the protease could be detected (Fig. S3). We next addressed whether C14 associates with AVRblb2 in planta by using confocal microscopy. As previously reported for the C14 ortholog RD21 (16, 17), fluorescently tagged full-length C14 and a C14 construct lacking the granulin domain (C14pep) localized to the endoplasmic reticulum (ER), endomembrane compartments, vacuoles, and apoplast (Fig. 2C and Fig. S4 B–E) and accumulated around haustoria in infected cells (Fig. S4F). Notably, in the presence of AVRblb2, the localization of C14 dramatically shifted toward the cell periphery, resulting in a marked overlap with the fluorescence signals of AVRblb2 and a plasma membrane marker (Fig. 2C and Fig. S5A). A similar shift in C14 distribution was observed in plant cells containing haustoria (haustoriated plant cells), resulting in colocalization and focal accumulation of AVRblb2 and C14 around haustoria (Fig. 2D). Altogether, these results indicate that AVRblb2 associates with C14 and alters its subcellular distribution.

AVRblb2 Prevents Secretion of the C14 Protease.

The increased peripheral accumulation of C14 triggered by AVRblb2 prompted us to address whether AVRblb2 affects secretion of C14. AVRblb2 significantly reduced apoplastic levels of C14 without affecting its intracellular accumulation (Fig. 3A and Fig. S5B). AVRblb2 did not inhibit secretion in general because it did not affect secretion of the pathogenesis-related serine protease P69B nor did it reduce overall protein levels in the apoplast (Fig. S5C). We confirmed these observations by microscopy. AVRblb2 significantly reduced the apoplastic accumulation of a C14pep:RFP fusion protein but increased its levels in the cytoplasm, mainly in the cell periphery and vacuoles (Fig. 3B). In contrast, AVRblb2 did not alter the apoplastic levels of a fusion of basic chitinase signal peptide to GFP (SP:GFP), confirming again that AVRblb2 does not generally prevent secretion (Fig. S5D). In summary, these results indicate that AVRblb2 inhibits secretion of the host protease C14.

Fig. 3.
AVRblb2 inhibits secretion of C14. (A) FLAG:Avrblb2 or FLAG:RFP was transiently coexpressed with C14 in N. benthamiana (two biological replicates for both FLAG:AVRblb2 and FLAG:RFP were used). Apoplastic and intracellular leaf extracts were separated ...

Consistent with these observations, we noted a gradual decrease in C14 levels in the tomato apoplast during P. infestans infection starting at 24 h after inoculation (Fig. 3C). Intracellular levels of C14 were also altered during infection with an increase in iC14 levels (Fig. 3C). These changes contrast sharply with the well-known increase in apoplastic levels of pathogenesis-related proteins, such as P69B, over the course of infection (Fig. 3C) (22). The decrease in apoplastic C14 during infection could be attributable to the preferential targeting of this protein to haustorial sites, where it is prevented from secretion. However, based on this experiment, we cannot exclude that the observed effect might involve additional effectors to AVRblb2.

AVRblb2 Localization Is Required for Its Virulence Function.

To address the link between AVRblb2 haustorial localization and its function, we tested several deletion mutants and identified an 8-aa C-terminal deletion mutant (AVRblb247–92) that did not exclusively accumulate at the cell periphery or around haustoria (Fig. 4 A–C). Notably, this mutant lost its ability to enhance P. infestans growth (Fig. 4D), weakly associated with C14 protease, and failed to attenuate apoplastic C14 accumulation (Fig. 4 E and F). Thus, localization of AVRblb2 and C14 secretion inhibition are genetically linked to the enhanced virulence activity of this effector. Conversely, the mutant retained its activity to trigger an Rpi-blb2–dependent hypersensitive response (Fig. 4G). These results indicate that AVRblb2 localization is essential for its virulence function but not for its avirulence activity.

Fig. 4.
An AVRblb2 mutant is impaired in haustorial localization and virulence effects but retains avirulence activity. (A) Overview of the constructs. The numbers correspond to AVRblb2 full-length protein amino acid residue positions. (B) Immunoblots of constructs ...

C14 Protease Positively Contributes to Immune Responses Against P. infestans.

To determine the extent to which C14 contributes to plant immunity, we altered C14 expression in N. benthamiana. Stable RNAi N. benthamiana lines, carrying two independent C14 hairpin constructs (5), showed significantly enhanced susceptibility to P. infestans compared with control lines as assessed by increased disease symptoms (Fig. S6). Conversely, overexpression of both C14 and C14pep in N. benthamiana resulted in reduced P. infestans colonization relative to controls (Fig. 5 and Fig. S7 A and B). This enhanced immunity conferred by C14 could be partially reversed by simultaneous overexpression of AVRblb2 (Fig. S7 C and D). In the course of these experiments, we also determined that C14 silencing did not affect Rpi-blb2 recognition of AVRblb2, indicating that C14 is not “guarded” by Rpi-blb2 (Fig. S8) (23, 24). These results indicate that C14 plays a positive role in plant immunity. We propose a model in which C14 is targeted by two classes of effectors in separate plant cell compartments: whereas AVRblb2 prevents secretion of C14 within haustoriated plant cells, other P. infestans effectors inhibit C14 in the apoplast as shown by Kaschani et al. (5) (Fig. 6).

Fig. 5.
A positive role for C14 in plant immunity against P. infestans. (A) Differential growth of P. infestans 88069 on tomato C14pep:RFP-expressing N. benthamiana lines (5 wk old). Pictures were taken at 8 d after infection. (B) Leaf apoplastic C14pep:RFP levels ...
Fig. 6.
Model of C14-EPIC-AVRblb2 interplay. C14 defense protease is secreted to the apoplast and inhibited by EPICs (5), which are secreted from growing P. infestans hyphae. Upon formation of haustoria, C14 is focally secreted to the extrahaustorial matrix. ...

A Plant Pathogen Effector That Probes Defense-Related Polarized Secretion.

In this study, we showed that the P. infestans effector AVRblb2 interferes with defense-related secretion in haustoriated plant cells. AVRblb2 can enhance susceptibility by reducing overall C14 levels in the host apoplast. The execution of plant cell-autonomous immunity requires cytoskeleton reorganization and polarized secretion (25, 26). Although some bacterial effectors are known to target secretory pathways, little is known about the underlying cellular and molecular processes (2729). The role of focal secretion in immunity has been difficult to dissect with standard genetic approaches because mutants often show pleiotropic effects that perturb plant development (26, 30). Our work indicates that effectors can be used as molecular probes to unravel unknown facets of focal immunity and potentially dissect the diversity of secretory vesicles and their cargo.

Our results implicate focal secretion of a plant defense protease in plant immunity. C14 could contribute to immunity by degrading non-self molecules or by playing a signaling role. C14 is known to accumulate during senescence and dehydration stress, but whether it also contributes to these processes is unclear (31, 32). During abiotic stress, C14 undergoes complex changes in subcellular localization. It accumulates in atypical ER-derived vesicles and, upon dehydration stress, can directly traffic into the vacuole in a nonclassical way bypassing the Golgi pathway (16, 17). Also, during desiccation, C14 is released to the cytosol and nucleus (32). We showed that perturbation of C14 trafficking by P. infestans AVRblb2 limits its role in plant immunity. AVRblb2 association with C14 might occur directly or indirectly through an intermediate transmembrane protein or molecule. AVRblb2 could intercept C14 at various subcellular sites, including during release or fusion of secretory vesicles to the plasma membrane at the haustorial interface. Finally, our findings that C14 overexpression overcomes AVRblb2 activity and leads to enhanced resistance to P. infestans point to immediate biotechnological applications for engineering late blight-resistant potato and tomato crops.

Materials and Methods

Plasmid Construction.

Effector expression constructs were designed to have N-terminal epitope or fluorescent tags replacing the native signal peptide to enable intracellular expression in plant cells. Detailed information about the plasmid constructs, transient gene-expression assays, and production of transgenic plants is described in SI Materials and Methods.

Co-IP Experiments.

FLAG:AVRblb2 and its plant interactors were coimmunoprecipitated with anti-FLAG resins under nondenaturing conditions. Co-IP experiments, preparation of peptides for LC-MS/MS, and Western blot analysis are described in SI Materials and Methods.

Secretion Inhibition Assays.

Secretion inhibition assays were performed by using Agrobacterium-mediated transient gene expression. Detailed procedures are provided in SI Materials and Methods.

Hypersensitive Response Cell-Death Assays.

Hypersensitive response assays on C14 silencing are described in SI Materials and Methods.

RT-PCR Assays.

Detailed RT-PCR procedures are explained in SI Materials and Methods.

Confocal Microscopy.

Confocal microscopy analysis was performed on Leica DM6000B/TCS SP5 confocal microscope (Leica Microsystems). Detailed procedures of confocal microscopy and callose/aniline blue staining of infected material is described in SI Materials and Methods.

Supplementary Material

Supporting Information:

Acknowledgments

We thank Ulla Bonas, Steve Whisson, and Frederick Boernke for providing biomaterials; Matthew Smoker, Liliya Serazetdinova, Jan Sklenář, and Richard O'Connell for technical advice and/or assistance; and Diane Saunders, Mireille van Damme, and Sylvain Raffaele for comments on drafts. This project was funded by the Gatsby Charitable Foundation; BASF Plant Science; Marie Curie Grant FP7-PEOPLE-2007-2-1-IEF; Deutsche Forschungsgemeinschaft Grants SCHO1347/1-1, HO 3983/7-1, and DAAD-HEC (Deutscher Akademischer Austausch Dienst and Higher Education Commission of Pakistan); and the Max Planck Society.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1112708109/-/DCSupplemental.

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