Fig 1.
The integration and expression of MoHrip1 and MoHrip2 in rice.
Different transgenic rice lines were chosen for molecular detection. (A). Schematic representation of the T-DNA region of pCXUN containing MoHrip1/MoHrip2-encoding genes. (B). Southern blotting of T1 transgenic rice. The numbers (1–13) represent MoHrip1-5, MoHrip1-9, MoHrip1-1, MoHrip1::HA-2, MoHrip1::HA-5, MoHrip1::HA-3, MoHrip1::HA-1, MoHrip2-5, MoHrip2-8, MoHrip2- 4, MoHrip2::HA-8, MoHrip2::HA-4, and MoHrip2::HA-4, respectively. M: DL15000 Marker. (C). Northern blotting of T2 transgenic rice. The numbers (1–8) represent MoHrip1-5, MoHrip1::HA-3, MoHrip1::HA-5, MoHrip1::HA-1, MoHrip2-8, MoHrip2-5, MoHrip2::HA-4, and MoHrip2::HA -8, respectively. The loading controls were 18S RNA and 28S rRNA. (D). Western blotting of the T2 transgenic rice. pCXUN was used as a negative control. (d1) The numbers (1–3) represent the empty vector pCXUN, MoHrip1::HA-1, MoHrip1::HA-5, respectively, and numbers 4 and 5 represent MoHrip1-5; (d2) The numbers (1–3) represent pCXUN, MoHrip2::HA -4, MoHrip2::HA -8, respectively, and the numbers 4 and 5 represent MoHrip2-5. All samples verified the expression of MoHrip1 and MoHrip2 in transgenic rice.
Table 1.
Primers used in this study.
Fig 2.
Disease symptoms on the leaves of MoHrip1, MoHrip2, pCXUN, and WT rice.
The detached leaves of two-week-old rice seedlings were sprayed with M. oryzae spores. The lowercase letters (a-f) represent MoHrip1-5 MoHrip1::HA-1, MoHrip1::HA-5 MoHrip2-5, MoHrip2::HA -4, and MoHrip2::HA -8, respectively. Representative leaves were photographed at 7 dpi. The results were obtained from three independent experiments.
Fig 3.
The Trypan blue staining of leaves challenged with M. oryzae spores.
The 2- and 6-dpi leaves were stained with Trypan blue solution to observe the development of disease symptoms at the inoculation sites. The arrows indicate the hyphae (2 dpi: bars = 100 μm; 6 dpi: bars = 50 μm).
Table 2.
Disease severity of rice blast in leaves of transgenic rice.
Fig 4.
The expression levels of defense-related genes in rice.
The WT, pCXUN, MoHrip1 and MoHrip2 rice were inoculated with blast suspension spores (2×105). At the indicated times, leaves were collected from these rice plants and measured via qRT-PCR. The relative expression of pathogenesis-related genes OsPR-1a (A) and OsPR-10a (B); SA signal-related genes OsEDS1 (C), OsNH1 (D), and OsPAL1 (E); and JA/ET biosynthesis-related genes OsLOX2 (F) and OsAOS2 (G) are shown. dpi: days post-inoculation; Error bars represent mean ± SD. Essentially identical results were obtained across three independent experiments. The asterisks indicate significant differences from the WT rice (*P < 0.05, **P < 0.01)
Fig 5.
The phenotype and ABA content of drought-stressed rice.
The MoHrip1-5 and MoHrip2-5 transgenic plants (line 5 of each transgenic plant) as well as the pCXUN rice and WT rice were well-watered for 2 weeks, and then water was discontinued for 16 days. The phenotype of rice before drought and after drought (A). The ABA content of transgenic and control rice on the 0 d, 6 d, 10 d, 14 d, and 16 d of drought stress (B). Essentially identical results were obtained across three independent experiments.
Fig 6.
The physiological indices of drought-stressed rice.
Analysis of RWC (A), WLR (B), WUE (C), and the chlorophyll content (D) in WT, pCXUN and transgenic rice under normal condition and after 14 d of drought stress. Error bars represent the mean ± SD of three replicates. Asterisks indicate significant differences from the WT rice (*P < 0.05, **P < 0.01).
Fig 7.
The expression levels of ABA-related genes in rice under drought-stress treatment.
RNA samples were prepared from rice leaves collected on the 0 d, 6 d, 10 d, 14 d, and 16 d of drought stress. The relative expression of OsNCED2 (A), OsNCED3 (B), OsZEP1 (C), and OsbZIP23 (D) is shown. Error bars represent mean ± SD. Essentially identical results were obtained across three independent experiments. The asterisks indicate significant differences from the WT rice (*P < 0.05, **P < 0.01)
Table 3.
Improved agronomic traits in transgenic rice expressing MoHrip1 or MoHrip2.