Disease symptoms on the leaves of <i>MoHrip1</i>, <i>MoHrip2</i>, <i>pCXUN</i>, and WT rice.

<p>The detached leaves of two-week-old rice seedlings were sprayed with <i>M</i>. <i>oryzae</i> spores. The lowercase letters (a-f) represent <i>MoHrip1</i>-5 <i>MoHrip1</i>::HA-1, <i>MoHrip1</i>::HA-5 <i>MoHrip2</i>-5, <i>MoHrip2</i>::HA -4, and <i>MoHrip2</i>::HA -8, respectively. Representative leaves were photographed at 7 dpi. The results were obtained from three independent experiments.</p

Primers used in this study.

<p>Primers used in this study.</p

Enhanced disease resistance and drought tolerance in transgenic rice plants overexpressing protein elicitors from <i>Magnaporthe oryzae</i>

<div><p>Exogenous application of the protein elicitors MoHrip1 and MoHrip2, which were isolated from the pathogenic fungus <i>Magnaporthe oryzae</i> (<i>M</i>. <i>oryzae</i>), was previously shown to induce a hypersensitive response in tobacco and to enhance resistance to rice blast. In this work, we successfully transformed rice with the <i>mohrip1</i> and <i>mohrip2</i> genes separately. The <i>MoHrip1</i> and <i>MoHrip2</i> transgenic rice plants displayed higher resistance to rice blast and stronger tolerance to drought stress than wild-type (WT) rice and the vector-control <i>pCXUN</i> rice. The expression of salicylic acid (SA)- and abscisic acid (ABA)-related genes was also increased, suggesting that these two elicitors may trigger SA signaling to protect the rice from damage during pathogen infection and regulate the ABA content to increase drought tolerance in transgenic rice. Trypan blue staining indicated that expressing MoHrip1 and MoHrip2 in rice plants inhibited hyphal growth of the rice blast fungus. Relative water content (RWC), water usage efficiency (WUE) and water loss rate (WLR) were measured to confirm the high capacity for water retention in transgenic rice. The <i>MoHrip1</i> and <i>MoHrip2</i> transgenic rice also exhibited enhanced agronomic traits such as increased plant height and tiller number.</p></div

The integration and expression of MoHrip1 and MoHrip2 in rice.

<p>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 <i>MoHrip1</i>-5, <i>MoHrip1</i>-9, <i>MoHrip1</i>-1, <i>MoHrip1</i>::HA-2, <i>MoHrip1</i>::HA-5, <i>MoHrip1</i>::HA-3, <i>MoHrip1</i>::HA-1, <i>MoHrip2</i>-5, <i>MoHrip2</i>-8, <i>MoHrip2</i>- 4, <i>MoHrip2</i>::HA-8, <i>MoHrip2</i>::HA-4, and <i>MoHrip2</i>::HA-4, respectively. M: DL15000 Marker. (C). Northern blotting of T₂ transgenic rice. The numbers (1–8) represent <i>MoHrip1</i>-5, <i>MoHrip1</i>::HA-3, <i>MoHrip1</i>::HA-5, <i>MoHrip1</i>::HA-1, <i>MoHrip2</i>-8, <i>MoHrip2</i>-5, <i>MoHrip2</i>::HA-4, and <i>MoHrip2</i>::HA -8, respectively. The loading controls were 18S RNA and 28S rRNA. (D). Western blotting of the T₂ transgenic rice. <i>pCXUN</i> was used as a negative control. (d1) The numbers (1–3) represent the empty vector <i>pCXUN</i>, <i>MoHrip1</i>::HA-1, <i>MoHrip1</i>::HA-5, respectively, and numbers 4 and 5 represent <i>MoHrip1</i>-5; (d2) The numbers (1–3) represent <i>pCXUN</i>, <i>MoHrip2</i>::HA -4, <i>MoHrip2</i>::HA -8, respectively, and the numbers 4 and 5 represent <i>MoHrip2</i>-5. All samples verified the expression of MoHrip1 and MoHrip2 in transgenic rice.</p

The phenotype and ABA content of drought-stressed rice.

<p>The <i>MoHrip1</i>-5 and <i>MoHrip2</i>-5 transgenic plants (line 5 of each transgenic plant) as well as the <i>pCXUN</i> 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.</p

Disease severity of rice blast in leaves of transgenic rice.

<p>Disease severity of rice blast in leaves of transgenic rice.</p

The physiological indices of drought-stressed rice.

<p>Analysis of RWC (A), WLR (B), WUE (C), and the chlorophyll content (D) in WT, <i>pCXUN</i> 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).</p

The Trypan blue staining of leaves challenged with <i>M</i>. <i>oryzae</i> spores.

<p>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).</p

The expression levels of ABA-related genes in rice under drought-stress treatment.

<p>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 <i>OsNCED2</i> (A), <i>OsNCED3</i> (B), <i>OsZEP1</i> (C), and <i>OsbZIP23</i> (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)</p