Fluorescent reporter analysis revealed the timing and localization of AVR-Pia expression, an avirulence effector of Magnaporthe oryzae

Abstract

In order to facilitate infection, the rice blast pathogen Magnaporthe oryzae secretes an abundance of proteins, including avirulence effectors, to diminish its host's defences. Avirulence effectors are recognized by host resistance proteins and trigger the host's hypersensitive response, which is a rapid and effective form of innate plant immunity. An understanding of the underlying molecular mechanisms of such interactions is crucial for the development of strategies to control disease. However, the expression and secretion of certain effector proteins, such as AVR-Pia, have yet to be reported. Reverse transcription-polymerase chain reaction (RT-PCR) revealed that AVR-Pia was only expressed during infection. Fluorescently labelled AVR-Pia indicated that AVR-Pia expression was induced during appressorial differentiation in the cells of both rice and onion, as well as in a penetration-deficient (Δpls1) mutant capable of developing melanized appressoria, but unable to penetrate host cells, suggesting that AVR-Pia expression is independent of fungal penetration. Using live-cell imaging, we also documented the co-localization of green fluorescent protein (GFP)-labelled AVR-Pia and monomeric red fluorescent protein (mRFP)-labelled PWL2, which indicates that AVR-Pia accumulates in biotrophic interfacial complexes before being delivered to the plant cytosol. Together, these results suggest that AVR-Pia is a cytoplasmic effector that is expressed at the onset of appressorial differentiation and is translocated to the biotrophic interfacial complex, and then into the host's cytoplasm.

Keywords: AVR-Pia; BIC, Pyricularia oryzae; appressorium; avirulence effector; rice.

Figure 1

Reverse transcription‐polymerase chain reaction (RT‐PCR) analysis of AVR‐Pia expression in in vitro and in planta Magnaporthe oryzae Ina168. (A) AVR‐Pia expression in liquid‐cultured Ina168 mycelia at 22 h post‐inoculation (hpi). (B) AVR‐Pia expression in Ina168‐infected rice leaf sheaths at various time points after inoculation. PC, positive control (AVR‐Pia DNA).

Figure 2

Quantification of Magnaporthe oryzae AVR‐Pia expression in compatible and incompatible rice plants. AVR‐Pia expression was quantified using quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) of RNA extracted from Ina168‐infected rice leaf sheaths at various time points after inoculation. AVR‐Pia levels are shown relative to actin expression, and the data points (n = 3) and error bars indicate means ± standard deviation.

Figure 3

AVR‐Pia promoter activity visualized using enhanced green fluorescence protein (eGFP) fluorescence in rice leaf sheath, onion epidermis and liquid culture. PPR::eGFP‐transformed Magnaporthe oryzae Ina168m95‐1 was used to monitor AVR‐Pia expression in rice leaf sheaths at 24 h post‐inoculation (hpi) (A) and 48 hpi (B), in onion epidermis at 24 hpi (C, D) and in liquid culture at 6 hpi (E) and 24 hpi (F). AP, appressorium; CD, conidium; IH, invasive hypha; white arrow, tip of hypha. Scale bars, 10 μm.

Figure 4

AVR‐Pia expression in a penetration‐deficient Magnaporthe oryzae mutant. Rice sheath assays of Δpls1‐PPR::eGFP (A) and Δpls1‐PPR::AVR‐Pia::4GGS::eGFP (C) were conducted at 24 h post‐inoculation (hpi), and fluorescence observation of liquid culture of Δpls1‐PPR::eGFP (B) and Δpls1‐PPR::AVR‐Pia::4GGS::eGFP (D) was performed at 24 hpi. AP, appressorium; GFP, green fluorescence protein; white arrow, tip of hypha. Scale bars, 5 μm.

Figure 5

Illustration of five enhanced green fluorescence protein (eGFP)‐fused AVR‐Pia constructs and the virulence of Magnaporthe oryzae transformed with each. The spacer sequence consisted of two, four or six glycine–glycine–serine repeats (2GGS, 4GGS and 6GGS, respectively) and was inserted between AVR‐Pia and eGFP. White arrows, native promoter region; spotted boxes, secreted signal; blue arrows, AVR‐Pia; latticed boxes, GGS spacer sequence; striped boxes, eGFP.

Figure 6

Preferential localization of enhanced green fluorescence protein (eGFP)‐labelled AVR‐Pia in Magnaporthe oryzae biotrophic interfacial complex (BIC) structures. White arrows indicate eGFP fluorescence. Scale bars, 10 μm. (A) The first invasive hyphae in the first invaded sheath epidermal cells at 27 and 30 h post‐inoculation (hpi) are indicated by eGFP‐labelled AVR‐Pia in the BIC‐like structures. (B) The third invasive hyphae in the third invaded sheath epidermal cells at 60 and 63 hpi are indicated by eGFP‐labelled AVR‐Pia in the BIC‐like structures. The boxes in the top right‐hand corners of the images are magnifications of the smaller corresponding boxes.

Figure 7

Co‐localization of AVR‐Pia and PWL2 in the biotrophic interfacial complex (BIC) structures of Magnaporthe oryzae. Microscopic observation of rice sheath cells infected with Ina168m95‐1‐pCSN43‐DEST‐PPR::AVR‐Pia::4GGS::eGFP‐pPWL2::PWL2::mRFP‐transformed M. oryzae was performed at 57 h post‐inoculation (hpi). IH, invasive hypha; white arrows, BICs. Scale bars, 10 μm. (A) Bright‐field image. (B) Green enhanced green fluorescence protein (eGFP)‐labelled AVR‐Pia fluorescence in BICs. (C) Red monomeric red fluorescent protein (mRFP)‐labelled PWL2 fluorescence in BIC. (D) Merged bright‐field, eGFP::AVR‐Pia (green) and mRFP::PWL2 (red) images.

Figure 8

Translocation of AVR‐Pia and PWL2 in the plasmolysed cytoplasm of Magnaporthe oryzae‐infected rice sheath cells at 61 h post‐inoculation (hpi). Asterisks, plasmolysed and concentrated rice cytoplasm. Scale bars, 10 μm. (A) Bright‐field image. (B) Green enhanced green fluorescence protein (eGFP)‐labelled AVR‐Pia fluorescence. (C) Red monomeric red fluorescent protein (mRFP)‐labelled PWL2 fluorescence. (D) Merged bright‐field, eGFP::AVR‐Pia (green) and mRFP::PWL2 image (red) images.