Moss Pathogenesis-Related-10 Protein Enhances Resistance to Pythium irregulare in Physcomitrella patens and Arabidopsis thaliana

Abstract

Plants respond to pathogen infection by activating signaling pathways leading to the accumulation of proteins with diverse roles in defense. Here, we addressed the functional role of PpPR-10, a pathogenesis-related (PR)-10 gene, of the moss Physcomitrella patens, in response to biotic stress. PpPR-10 belongs to a multigene family and encodes a protein twice the usual size of PR-10 proteins due to the presence of two Bet v1 domains. Moss PR-10 genes are differentially regulated during development and inoculation with the fungal pathogen Botrytis cinerea. Specifically, PpPR-10 transcript levels increase significantly by treatments with elicitors of Pectobacterium carotovorum subsp. carotovorum, spores of B. cinerea, and the defense hormone salicylic acid. To characterize the role of PpPR-10 in plant defense against pathogens, we conducted overexpression analysis in P. patens and in Arabidopsis thaliana. We demonstrate that constitutive expression of PpPR-10 in moss tissues increased resistance against the oomycete Pythium irregulare. PpPR-10 overexpressing moss plants developed less symptoms and decreased mycelium growth than wild type plants. In addition, PpPR-10 overexpressing plants constitutively produced cell wall depositions in protonemal tissue. Ectopic expression of PpPR-10 in Arabidopsis resulted in increased resistance against P. irregulare as well, evidenced by smaller lesions and less cellular damage compared to wild type plants. These results indicate that PpPR-10 is functionally active in the defense against the pathogen P. irregulare, in both P. patens and Arabidopsis, two evolutionary distant plants. Thus, P. patens can serve as an interesting source of genes to improve resistance against pathogen infection in flowering plants.

Keywords: Arabidopsis thaliana; Physcomitrella patens; Pythium irregulare; cell wall; defense; pathogenesis-related protein 10.

FIGURE 1

Alignments of deduced amino acid sequences of PpPR-10 with other moss PR-10-like proteins. Alignments were performed with ClustalW. PpPR-10 (Phypa_105033) used in this study is highlighted (blue underline). Red lines and the green asterisk mark the GXGXXG motif and the conserved lysine (K68), respectively.

FIGURE 2

Subcellular localization of PpPR-10 in agroinfiltrated tobacco leaves. Confocal microscopy images of tobacco leaves agroinfiltrated with the 35S:PpPR-10-GFP construct. Leaves have been imaged 2 days after agroinfiltration by confocal microscopy. Red fluorescence belongs to chlorophyll in chloroplasts (A), green fluorescence belongs to GFP (B), and merged fluorescences are shown in (C). (D) Immunoblot detection of PpPR-10-GFP fusion protein (red arrow) in agroinfiltrated tobacco leaves, showing that the signal is mostly derived from the fusion protein. Ten micrograms of total soluble proteins were separated in SDS-PAGE and Western blot analysis was performed using an antibody for GFP. The 27 kDa protein corresponds to cleaved GFP (black arrow). The scale bars represent 20 μm.

FIGURE 3

PpPR-10 expression in response to pathogens and hormones. (A) Expression of PpPR-10 in response to elicitors of P. wasabiae (P. w) and P.c. carotovorum (P.c.c), spores of B. cinerea and mycelium of P. irregulare at different hours after treatments. As control colonies were treated with water. (B) Expression of PpPR-10 in moss colonies treated with SA, MeJA, ABA, and auxin 1-naphthalene acetic acid (NAA). Ten micrograms of total RNA were separated on formaldehyde–agarose gels, transferred to a nylon membrane and hybridized to a ³²P-labeled PpPR-10 cDNA probe. Ethidium bromide staining of rRNA was used to ensure equal loading of RNA samples. Experiments were repeated thrice with similar results.

FIGURE 4

Generation of PpPR-10 overexpression P. patens plants. (A) Schematic representation of PpPR-10 overexpressing construct using plasmid pTHUbi. (B) Transcript levels of PpPR-10 in untreated (Control) and Pectobacterium corotovorum subsp. carotovorum (P.c. c) elicitor-treated wild type plants, and untreated pUBI:PpPR-10 overexpressing lines. (C) Phenotype of wild type, pUBI:PpPR-10-1, and pUBI:PpPR-10-3 moss colonies. (D) Size of wild-type, pUBI:PpPR-10-1, and pUBI:PpPR-10-3 moss colonies grown for 21 days in BCDAT medium measured as diameter in centimeters. Results and standard deviation correspond to 16 colonies per sample. Asterisk for pUBI:PpPR-10-3 colonies indicates that the values are significantly different from wild type plants according to Kruskal–Wallis test: P < 0.001.

FIGURE 5

Symptom development and mycelium growth of P. irregulare in wild type and PpPR-10 overexpressing moss lines. (A) Symptom development in wild type, pUBI:PpPR-10-1 and pUBI:PpPR-10-3 moss colonies. (B) P. irregulare DNA levels were estimated by qPCR analysis. Ratios of P. irregulare to P. patens (Pp) gDNA were determined by qPCR with primers ITSf/ITSr and EFf/EFr, respectively. The results and standard deviation of three independent triplicate experiments are shown. (C) Measurement of cell death by Evans blue staining 24 h after inoculation of wild type, pUBI:PpPR-10-1 and pUBI:PpPR-10-3 moss colonies with P. irregulare. Data were expressed as the optical density (OD) at 600 nm per milligram of dry weight (DW). Values are means with standard deviations of six independent replicate moss samples. Experiments were repeated trice with similar results. Asterisks indicate a statistically significant difference between the wild type and overexpressing PpPR-10 plants [Students t-test, P < 0.005 (^∗)]. The scale bars represent 0.5 cm.

FIGURE 6

Cell wall associated modification in overexpressing PpPR-10 P. patens protonemal tissues. Untreated protonemal tissues of wild type (A), pUBI:PpPR-10-1 (B), and pUBI:PR-10-3 (C) stained with solophenyl flavine. Untreated protonemal tissues stained with methyl blue showing callose deposition in wild type (D), pUBI:PpPR-10-1 (E), and pUBI:PR-10-3 (F). The scale bars represent 20 μm.

FIGURE 7

Overexpression of PpPR-10 in Arabidopsis. (A) Representative semiquantitative RT-PCR analysis of PpPR-10 transcript levels in Arabidopsis PpPR-10 transformed lines. The ubiquitin gene AT3G52590 was used as an internal control. (B) Immunoblot detection of PpPR-10-GFP fusion protein (red arrow) in transgenic Arabidopsis lines At 35S:PpPR-10-1 (#1), At 35S:PR-10-2 (#2) and At 35S:PR-10-5 (#5). Ten micrograms of total soluble proteins were separated in SDS-PAGE and Western blot analysis was performed using an antibody for GFP. As a control for GFP detection, a protein sample from transgenic Arabidopsis plants expressing constitutively unfused GFP (35S-GFP) was included. The 27 kDa protein corresponds to cleaved GFP (black arrow). (C) Fluorescent microscopy of leaves and roots of wild type (Col-0), At 35S:PpPR-10-1 (#1), At 35S:PR-10-2 (#2) and At 35S:PR-10-5 (#5) Arabidopsis lines. The scale bars represent 50 μm.

FIGURE 8

Symptom development and ion leakage measurements in wild type and Arabidopsis PpPR-10 overexpressing lines. (A) Symptom development in wild type, At 35S:PR-10-1, At 35S:PR-10-2, and At 35S:PR-10-5 leaves. (B) Electrolyte leakage in wild type and overexpressing PpPR-10 Arabidopsis plants.

FIGURE 9

Pythium irregulare infected Arabidopsis wild type and overexpressing PpPR-10 lines. Symptom development in 2 days-inoculated Arabidopsis leaves of wild type (A), At 35S:PpPR-10-1 (B), At 35S:PR-10-2 (C), and At 35S:PR-10-5 (D). Red arrows indicate the border of the lesions. Hyphae in the infected tissues were visualized using the fluorescent dye solophenyl flavine in wild type (E), At 35S:PpPR-10-1 (F), At 35S:PR-10-2 (G), and At 35S:PR-10-5 (H). Magnification of the same pictures as in (A–G) are shown in (I–P), respectively. Scale bars represent in (A–H); 1 mm and in (I–P); 0,3 mm.