Transgenic rice expressing Allium sativum leaf agglutinin (ASAL) exhibits high-level resistance against major sap-sucking pests

<p>Abstract</p> <p>Background</p> <p>Rice (<it>Oryza sativa</it>) productivity is adversely impacted by numerous biotic and abiotic factors. An approximate 52% of the global production of rice is lost annually owing to the damage caused by biotic factors, of which ~21% is attributed to the attack of insect pests. In this paper we report the isolation, cloning and characterization of <it>Allium sativum </it>leaf agglutinin (<it>asal</it>) gene, and its expression in elite indica rice cultivars using <it>Agrobacterium</it>-mediated genetic transformation method. The stable transgenic lines, expressing ASAL, showed explicit resistance against major sap-sucking pests.</p> <p>Results</p> <p><it>Allium sativum </it>leaf lectin gene (<it>asal</it>), coding for mannose binding homodimeric protein (ASAL) from garlic plants, has been isolated and introduced into elite indica rice cultivars susceptible to sap-sucking insects, viz., brown planthopper (BPH), green leafhopper (GLH) and whitebacked planthopper (WBPH). Embryogenic calli of rice were co-cultivated with <it>Agrobacterium </it>harbouring pSB111 super-binary vector comprising garlic lectin gene <it>asal </it>along with the herbicide resistance gene <it>bar</it>, both under the control of CaMV35S promoter. PCR and Southern blot analyses confirmed stable integration of transgenes into the genomes of rice plants. Northern and western blot analyses revealed expression of ASAL in different transgenic rice lines. In primary transformants, the level of ASAL protein, as estimated by enzyme-linked immunosorbent assay, varied between 0.74% and 1.45% of the total soluble proteins. <it>In planta </it>insect bioassays on transgenic rice lines revealed potent entomotoxic effects of ASAL on BPH, GLH and WBPH insects, as evidenced by significant decreases in the survival, development and fecundity of the insects.</p> <p>Conclusion</p> <p><it>In planta </it>insect bioassays were carried out on <it>asal </it>transgenic rice lines employing standard screening techniques followed in conventional breeding for selection of insect resistant plants. The ASAL expressing rice plants, bestowed with high entomotoxic effects, imparted appreciable resistance against three major sap-sucking insects. Our results amply demonstrate that transgenic indica rice harbouring <it>asal </it>exhibit surpassing resistance against BPH, GLH and WBPH insects. The prototypic <it>asal </it>transgenic rice lines appear promising for direct commercial cultivation besides serving as a potential genetic resource in recombination breeding.</p

Transgenic pearl millet male fertility restorer line (ICMP451) and hybrid (ICMH451) expressing Brassica juncea Nonexpressor of pathogenesis related genes 1 (BjNPR1) exhibit resistance to downy mildew disease.

Brassica juncea Nonexpressor of pathogenesis-related genes 1 (BjNPR1) has been introduced into pearl millet male fertility restorer line ICMP451 by Agrobacterium tumefaciens-mediated genetic transformation. Transgenic pearl millet plants were regenerated from the phosphinothricin-resistant calli obtained after co-cultivation with A. tumefaciens strain LBA4404 harbouring Ti plasmid pSB111-bar-BjNPR1. Molecular analyses confirmed the stable integration and expression of BjNPR1 in transgenic pearl millet lines. Transgenes BjNPR1 and bar were stably inherited and disclosed co-segregation in subsequent generations in a Mendelian fashion. Transgenic pearl millet hybrid ICMH451-BjNPR1 was developed by crossing male-sterile line 81A X homozygous transgenic line ICMP451-BjNPR1. T3 and T4 homozygous lines of ICMP451-BjNPR1 and hybrid ICMH451-BjNPR1 exhibited resistance to three strains of downy mildew pathogen, while the untransformed ICMP451 and the isogenic hybrid ICMH451 plants were found susceptible. Following infection with S. graminicola, differential expression of systemic acquired resistance pathway genes, UDP-glucose salicylic acid glucosyl transferase and pathogenesis related gene 1 was observed in transgenic ICMP451-BjNPR1 and untransformed plants indicating the activation of systemic acquired resistance pathway contributing to the transgene-mediated resistance against downy mildew. The transgenic pearl millet expressing BjNPR1 showed resistance to multiple strains of S. graminicola and, as such, seems promising for the development of durable downy mildew resistant hybrids

Microscopic view of <i>S. graminicola</i> strain <i>Sg</i> 384 infected leaf surface of transgenic ICMP451-<i>BjNPR1</i> and untransformed ICMP451.

<p>(A) Untransformed ICMP451 leaf showing mycelial growth on leaf surface. (B) Transgenic ICMP 451-<i>BjNPR1</i> leaf surface showing healthy growth without any mycelia.</p

Fungal bioassays on isogenic hybrids ICMH451 (81A x untransformed ICMP 451) and ICMH451-<i>BjNPR1</i> (81A x ICMP 451- <i>BjNPR1</i>) challenged with <i>S. graminicola</i> strains <i>Sg</i> 445, <i>Sg</i> 492 and <i>Sg</i> 384.

<p>ICMH 451-<i>BjNPR1</i> plants exhibiting resistance with healthy and normal growth. ICMH 451 plants exhibiting susceptibility with stunted growth and chlorotic symptoms.</p

Southern and northern blot analyses of putative ICMP 451- <i>BjNPR1</i> transgenics of pearl millet in T₀ generation.

<p>(A) Southern blot of <i>Bgl</i>II digested genomic DNA probed with <i>BjNPR1</i> sequence showing hybridized bands of >3.0 kbp. (B) Northern blot probed with <i>BjNPR1</i> internal sequence showing hybridizable bands of ∼2 kb. Lane UC: Untransformed ICMP 451. Lanes 1&2: Pearl millet <i>BjNPR1</i> transgenics 1T₀ and 2T₀, respectively.</p

Sub-cellular localization of GFP/ BjNPR1-GFP fusion protein before and after salicylic acid treatment in pearl millet.

<p>(A and B) Leaf sheath cells transiently expressing GFP showing green fluorescence both in the cytoplasm and nucleus, before and after salicylic acid treatment, respectively. (A1) Bright field image. (A2) Overlay image. (B1) Bright field image (B2) Overlay image. (C) Leaf sheath cells transiently expressing BjNPR1-GFP showing green fluorescence in the cytoplasm before salicylic acid treatment. (C1) Bright field image. (C2) Overlay image. (D) Leaf sheath cells transiently expressing BjNPR1-GFP showing green fluorescence in the nucleus after salicylic acid treatment. (D1) Bright field image. (D2) Overlay image.</p

Fungal bioassays for downy mildew resistance in isogenic hybrids ICMH451 and ICMH451-<i>BjNPR1.</i>

<p>Fungal bioassays for downy mildew resistance in isogenic hybrids ICMH451 and ICMH451-<i>BjNPR1.</i></p

ICMP 451-<i>BjNPR1</i> transgenics exhibiting tolerance and untransformed ICMP 451 exhibiting sensitivity to 400µM salicylic acid.

<p>(A) Seeds of ICMP 451-<i>BjNPR1</i> transgenics and untransformed ICMP 451showing normal germination on MS medium without salicylic acid. (B) Seeds of ICMP 451-<i>BjNPR1</i> transgenics showing normal germination on MS medium supplemented with 400 µM salicylic acid, while untransformed ICMP 451 failed to germinate.</p

Randomly selected 2T₁ plants of ICMP 451-<i>BjNPR1</i> showing co-segregation of Basta tolerance, amplification of <i>bar</i> and <i>BjNPR1</i> genes, and expression of <i>BjNPR1</i> transcripts.

<p>(A) Basta leaf dip assay showing segregation for Basta tolerance and susceptibility. (B) PCR analysis showing segregation for <i>bar</i> gene. (C) PCR analysis showing segregation for <i>BjNPR1</i> gene. (D) Northern blot showing segregation for expression of <i>BjNPR1</i>. Lane M: 1 kb DNA molecular weight marker. Lane UC: Untransformed ICMP 451. Lanes 1 to 11: Randomly selected T₁ plants of 2T₁<i>BjNPR1</i>transgenics.</p

Normalized transcript values of <i>PR1</i> and <i>SAGT</i> in the Untransformed ICMP 451 and transgenic ICMP 451- <i>BjNPR1</i> challenged with <i>S. graminicola</i> strain, <i>Sg</i> 384.

<p>Normalized transcript values of <i>PR1</i> and <i>SAGT</i> in the Untransformed ICMP 451 and transgenic ICMP 451- <i>BjNPR1</i> challenged with <i>S. graminicola</i> strain, <i>Sg</i> 384.</p