SP-LL-37, human antimicrobial peptide, enhances disease resistance in transgenic rice

<div><p>Human LL-37 is a multifunctional antimicrobial peptide of cathelicidin family. It has been shown in recent studies that it can serve as a host’s defense against influenza A virus. We now demonstrate in this study how signal peptide LL-37 (SP-LL-37) can be used in rice resistance against bacterial leaf blight and blast. We synthesized LL-37 peptide and subcloned in a recombinant pPZP vector with pGD1 as promoter. SP-LL-37 was introduced into rice plants by <i>Agrobacterium</i> mediated transformation. Stable expression of SP-LL-37 in transgenic rice plants was confirmed by RT-PCR and ELISA analyses. Subcellular localization of SP-LL-37-GFP fusion protein showed evidently in intercellular space. Our data on testing for resistance to bacterial leaf blight and blast revealed that the transgenic lines are highly resistant compared to its wildtype. Our results suggest that LL-37 can be further explored to improve wide-spectrum resistance to biotic stress in rice.</p></div

Generation and molecular analysis of the transgenic lines expressing pPZP::SP-LL-37.

<p>(A) TaqMan PCR analysis for copy number assays using TaqMan probe for selected homozygous T₁ plants; T₂ homo; T₂-homozygous, T₂ hetero; T₂-heterozygous, NT; no template, WT; wild type, 6–1~20–3; 19 T₁ plants. (B) SP-LL-37 gene expression in T₁ homo transgenic lines using RT-PCR. Total RNA was isolated from each plant, and 0.5 <i>μg</i> of this RNA was amplified with SP-LL-37-specific primers. Rice actin gene was amplified as a loading control. (C) ELISA analysis of SP-LL-37 lines in bovine sperm conditioned media collected at T₁-transgenic rice plants.</p

<i>In planta</i> bioassay of <i>M</i>. <i>oryzae</i> (10⁵ spores/ml) on transgenic lines.

<p><b>(A)</b> Whole plant infection assay in WT and T₂ transgenic homo lines. <b>(B)</b> Blast infected leaves of WT and T₂ transgenic homo lines. Infected leaf area was measured in % DLA (Diseased leaf area) at 10 dpi. The data are presented as means ± SEM from three independent estimations.</p

Identification of pPZP::SP-LL-37 insertion transgenic plants.

<p>The genomic structures of insertion alleles were determined by FST analysis in which boxes, bold lines, and triangles indicate exons, intron, and pPZP::SP-LL-37, respectively. The arrow and arrowhead indicate gene specific primer pairs from genomic DNA. Genomic DNA was isolated from the leaves of pPZP::SP-LL-37 regenerated plants for PCR analysis. WT: wild type, T: T₀ plant with single T-DNA insert for pPZP::SP-LL-37 insertion line.</p

Analysis of flanking regions adjacent to the left or right border of T-DNA.

<p>(A) The diagram illustrates the PCR strategy used to obtain sequences flanking the T-DNA insertion sites. Restriction enzymes (shown as lightning) with recognition sites near the T-DNA border sequences (LB and RB) were used to digest DNA from the T-DNA insertion lines. Enzymes cut both within the T-DNA, and ligations were performed to circularize purified digestion products with <i>Bfa</i> I adapter. PCR1 using primers (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0172936#pone.0172936.s001" target="_blank">S1 Table</a>) specific to adapter and T-DNA (AP1+R1 for RB analysis or AP1 + Fa1 for LB analysis). PCR2 using nested primer pairs (AP2+R2 for RB analysis or AP2+Fa2 for LB analysis). (B) PCR2 products were separated by gel electrophoresis. The first lane of each row contains a molecular marker (size of bottom five bands: 200, 400, 600, 800 and 1,000 nt).</p

Identification and characterization of the T2 homozygous plants.

<p><b>(A)</b> Phenotype of transgenic T₂ generation in pot. <b>(B)</b> Whole plant infection assay of WT and T₂ generation. Disease development in T₂ generation inoculated with <i>X</i>. <i>oryzae</i> KACC10859 strain. <b>(C)</b> Disease scoring was conducted 10 days after inoculation. WT; Dongjinbyo rice, 1~5; T₂ transgenic homo lines.</p

Subcellular localization of the SP-LL-37 protein in tobacco.

<p>GFP protein was attached to the end of C-terminal of LL-37 protein to see the localization in tobacco cells. <b>(A)</b> Cartoon image of constructs used in subcellular localization. <b>(B)</b> Fluorescence image of cell membrane (<i>N</i>. <i>benthamiana</i> epidermal cells) from <i>Agrobacterium</i>-mediated transient expression (using the p19 protein to enhance the expression level) and cytoplasm expressing the SP-LL-37 protein with positive and negative control by confocal microscopy.</p

Frequencies of T-DNA insertions in the genic and intergenic regions.

<p>Frequencies of T-DNA insertions in the genic and intergenic regions.</p

Copy number frequency of pPZP::SP-LL-37 in transgenic plants.

<p>Copy number frequency of pPZP::SP-LL-37 in transgenic plants.</p

Development of Self-Compatible <em>B. rapa</em> by RNAi-Mediated <em>S</em> Locus Gene Silencing

<div><p>The self-incompatibility (SI) system is genetically controlled by a single polymorphic locus known as the <em>S</em>-locus in the Brassicaceae. Pollen rejection occurs when the stigma and pollen share the same <em>S</em>-haplotype. Recognition of <em>S</em>-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (<em>SRK</em>) and <em>S</em>-locus protein 11 or <em>S</em> locus Cysteine-rich (<em>SP11/SCR</em>) protein. Here, we examined the function of <em>S₆₀,</em> one <em>SP11/SCR</em> allele of <em>B. rapa</em> cv. Osome, using a RNAi-mediated gene silencing approach. The transgenic RNAi lines were highly self-compatible, and this trait was stable in subsequent generations, even after crossing with other commercial lines. These findings also suggested that the resultant self-compatibility could be transferred to commercial cultivars with the desired performances in <em>B. rapa</em>.</p> </div