Purification and characterization of a novel hypersensitive response-inducing elicitor from Magnaporthe oryzae that triggers defense response in rice

Abstract

Background: Magnaporthe oryzae, the rice blast fungus, might secrete certain proteins related to plant-fungal pathogen interactions.

Methodology/principal findings: In this study, we report the purification, characterization, and gene cloning of a novel hypersensitive response-inducing protein elicitor (MoHrip1) secreted by M. oryzae. The protein fraction was purified and identified by de novo sequencing, and the sequence matched the genomic sequence of a putative protein from M. oryzae strain 70-15 (GenBank accession No. XP_366602.1). The elicitor-encoding gene mohrip1 was isolated; it consisted of a 429 bp cDNA, which encodes a polypeptide of 142 amino acids with a molecular weight of 14.322 kDa and a pI of 4.53. The deduced protein, MoHrip1, was expressed in E. coli. And the expression protein collected from bacterium also forms necrotic lesions in tobacco. MoHrip1 could induce the early events of the defense response, including hydrogen peroxide production, callose deposition, and alkalization of the extracellular medium, in tobacco. Moreover, MoHrip1-treated rice seedlings possessed significantly enhanced systemic resistance to M. oryzae compared to the control seedlings. The real-time PCR results indicated that the expression of some pathogenesis-related genes and genes involved in signal transduction could also be induced by MoHrip1.

Conclusion/significance: The results demonstrate that MoHrip1 triggers defense responses in rice and could be used for controlling rice blast disease.

Figure 1. The purification of MoHrip1 from M. oryzae.

A. Concentrated culture filtrate was loaded on HiTrap™ Q HP 5 ml column at a flow rate of 2 ml/min. Four peaks were collected, and the target protein was included in peak a2. B. Peak a2 was pooled and applied to native-PAGE and electroelution. The b3 fraction was the target elicitor protein. C. SDS-PAGE analysis of the purified elicitor protein, MoHrip1, showing a single band with Coomassie Brilliant Blue R-250 staining. (1: MoHrip1, M: protein molecular weight marker).

Figure 2. The hypersensitive response induced by MoHrip1 in tobacco leaves.

A. The response was photographed 24 hours after injection. (a) The MoHrip1 treatment (30 µM), (b) Control treatment with Mes-NaOH buffer (20 mM). B. Series of concentration-induced HR activity. a: 30 µM Bovine serum albumin (BSA) b-h: 30 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, 0.1 µM and 0.01 µM MoHrip1, respectively. The elicitor protein was dissolved in sterile distilled water in series of concentrations. The minimum concentration of MoHrip1 that induced HR activity was 5 µM. C. Tobacco leaves with control treatment (20 mM) stained by trypan blue. D. Tobacco leaves with MoHrip1 treatment (30 µM) stained by trypan blue. Dead tobacco leaf cells, which are the hallmark of HR, were stained by trypan blue. Scale bar = 10 µM.

Figure 3. Purification of recombinant MoHrip1.

M: protein molecular weight marker, 1: Purified His-tagged MoHrip1, 2: Total E. coli expressed proteins.

Figure 4. ROS burst in tobacco after MoHrip1 treatment.

A. Microscopic observation of H₂O₂ accumulation in tobacco leaves. (a) Tobacco leaves treated with sterile distilled water, (b) MoHrip1-treated leaves. H₂O₂ accumulation (as indicated by diaminobenzidine staining) appeared in the veins and stomata of elicitor-treated leaves but not in leaves treated with sterile distilled water. Scale bar = 50 µm. B. ROS formation in tobacco cell culture after elicitor treatment, flg22 treatment and sterile distilled water treatment was detected in 96-well plates by chemiluminescence. ROS formation in both the MoHrip1-treated and flg22-treated cell cultures reached a maximum at approximately 5 min and declined thereafter to the level of the negative control. Each data point represents three replicates. Error bars represent ± SD of the mean.

Figure 5. MoHrip1-induced callose deposition and extracellular medium alkalinization in tobacco.

A. Callose deposition in tobacco leaves. Tobacco leaves were infiltrated with MoHrip1 (50 µL of a 5 µM solution of MoHrip1) or Mes-NaOH buffer (20 mM, pH 6.0) as a control. After a 24 hour incubation period, the tobacco leaves were bleached to remove their chlorophyll and then stained with aniline blue. The samples were observed under bright-field (a and c) and UV fluorescence (b and d) microscopy. Apparent punctiform callose deposits around the cell wall were photographed in the MoHrip1-treated leaves (b). a and b: MoHrip1-treated leaves, c and d: control. Scale bar = 10 µM. B. The kinetics of the extracellular medium alkalinization induced by MoHrip1 in tobacco suspension. A distinct pH increase in the elicitor-treated cell culture was monitored for the first 10 minutes, and the pH stabilized after 100 minutes. Each data point represents three replicates. The error bars represent ± SD of the mean.

Figure 6. Representative disease symptoms on leaves of the MoHrip1-treated and control rice seedlings.

Rice seedlings were sprayed with MoHrip1 (5 µM) or Mes-NaOH (20 mM) as a control 3 days before being treated with M. oryzae spores. The leaves of representative plants were photographed 7 days post-inoculation.

Figure 7. ROS generation induced by MoHrip1 in rice.

A H₂O₂ burst was obviously elicited by MoHrip1 (dissolved in sterile distilled water) or flg22 (positive control, dissolved in sterile distilled water) within 10 minutes in rice leaves, but no H₂O₂ burst was detected in the samples treated with sterile distilled water (negative control). After 20 minutes, the level of ROS production in MoHrip1- or flg22-treated samples decreased to that in the negative control samples. The error bars represent ± SD of the mean. Essentially identical results were obtained in three independent experiments.

Figure 8. Defense-related gene expression induced by MoHrip1 in rice leaves.

PR genes were induced and their expression persisted for days in M. oryzae strain RO1-1-treated plants and in MoHrip1-pretreated plants that were not exposed to M. oryzae strain RO1-1. In contrast, the expression of PR genes was induced after 2 days of treatment and declined thereafter in the RO1-1-treated samples. The error bars represent ± SD of the mean. Essentially identical results were obtained in three independent experiments. A. Expression of OsPR-1a induced by MoHrip1 in rice leaves, B. Expression of OsPR-10a induced by MoHrip1 in rice leaves.

Figure 9. Certain marker genes of the defense signaling pathway are involved in the effects of MoHrip1 on rice.

A. OsEDS1, B. OsPAL1, C. OsNH1, D. OsLOX2, E. OsAOS2. Marker genes for the SA-dependent defense pathway (OsEDS1, OsPAL1 and OsNH1) were all significantly induced by MoHrip1 on the second day after treatment, and the same expression pattern was found for the JA/Et pathway gene OsLOX2, but strong induction was not observed for OsAOS2 (another JA/Et signaling pathway marker gene). Dpi: days post-inoculation. Error bars represent ± SD of the mean. Essentially identical results were obtained in three independent experiments.