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.

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 was not affected by plasmolysis and overlapped with a plasma membrane marker (pm-RK). (B) P. infestans-infected cells (red) focally accumulated GFP:AVRblb2 (green) around haustoria (arrowheads). Magenta represents the signal from plastids. (C) GFP:Avrblb2 transgenic N. benthamiana plants (5 wk old) were more susceptible to infection and enabled faster P. infestans sporulation compared with controls. (D) Quantitative scoring of infection stages and GFP expression intensities on WT, control (GFP), and GFP:Avrblb2 transgenic lines. The x axis represents the position on the intensity transect, and the white line represents GFP signal intensities of transgenic lines. The y axis depicts the number of successful infections (each leaf is inoculated with Phytophthora at six different spots).

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 alone or with C14 in N. benthamiana. Immunoprecipitates obtained with anti-FLAG antiserum and total protein extracts were immunoblotted with appropriate antisera. (C) C14pep:RFP was detected in vacuoles and as apoplastic aggregates, which did not colocalize with plasma membrane CFP (pm:CFP; Upper). Coexpression of GFP:Avrblb2 increased C14pep:RFP intensity at the plasma membrane (Lower). Fluorescence intensities of CFP/GFP/RFP in membrane transects (yellow arrowheads) at 3 d postinfiltration (dpi) are illustrated. (D and E) C14:GFP (D) and C14pep:GFP (E) accumulate at haustorial sites (arrowheads). Accumulation was enhanced upon RFP:Avrblb2 coexpression. Pictures were taken at 3 d post infection (D) and 4 d post infection (E).

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 and stained with Coomassie Brilliant Blue (CBB). Immunoblots with appropriate antisera showed reduced apoplastic iC14 (i) and mC14 (m) levels. (B) Confocal sectioning of epidermal cells revealed that apoplastic accumulation of C14pep:RFP (Upper) was reduced and shifted to intracellular vacuoles (Lower) upon transient coexpression with GFP:Avrblb2. (C) P. infestans colonization of tomato is associated with a decrease in apoplastic C14. Apoplastic or intracellular C14 and P69B protein levels were assessed from infected tomato leaves over a time course. Immunoblotting of apoplastic fluids and intracellular protein extracts showed a marked reduction of apoplastic C14 levels starting at 24 h after inoculation, whereas intracellular iC14 levels increased. In contrast, a significant increase in both apoplastic and intracellular P69B levels were observed.

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 expressed in N. benthamiana. (C) GFP:AVRblb2^47–100 (GFP:AVRblb2) shows localization to the cell periphery in uninfected leaves and focal accumulation around haustoria (arrowheads) in leaves infected with P. infestans 88069, whereas the mutant GFP:AVRblb2^47–92 shows a subcellular distribution that resembles GFP (nucleocytoplasmic) and does not focally accumulate at haustoria at 3 d post infection. (Scale bars: 25 μm.) Green color is GFP, and magenta is plastid fluorescence. (D) AVRblb2 mutant does not enhance pathogen growth. (E) Co-IP of GFP:AVRblb2, GFP:AVRblb2^47–92, and GFP with C14. (F) Levels of apoplastic C14 are reduced by coexpression of AVRblb2 but not by GFP:AVRblb2^47–92 or GFP. (G) GFP:AVRblb2^47–92 retains avirulence activity. A. tumefaciens-mediated transient expression of GFP-fused Avrblb2 constructs or control (GFP) in Rpi-blb2 transgenic and WT N. benthamiana.

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 in descendants of two independent transgenic N. benthamiana lines were measured by using confocal microscopy. (C) Plants with apoplastic C14pep:RFP accumulation showed reduced hyphal growth of P. infestans 88069 compared with ER-GFP–expressing control N. benthamiana lines (5 wk old). Growth efficiency was plotted as average total growing necrosis diameter (n = 6) at 5 d after infection.

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. The RXLR effector AVRblb2 is translocated from P. infestans hyphae into the host cells and prevents secretion of C14.