Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper.

Functional characterization of a defensin, J1-1, was conducted to evaluate its biotechnological potentiality in transgenic pepper plants against the causal agent of anthracnose disease, Colletotrichum gloeosporioides. To determine antifungal activity, J1-1 recombinant protein was generated and tested for the activity against C. gloeosporioides, resulting in 50% inhibition of fungal growth at a protein concentration of 0.1 mg·mL-1. To develop transgenic pepper plants resistant to anthracnose disease, J1-1 cDNA under the control of 35S promoter was introduced into pepper via Agrobacterium-mediated genetic transformation method. Southern and Northern blot analyses confirmed that a single copy of the transgene in selected transgenic plants was normally expressed and also stably transmitted to subsequent generations. The insertion of T-DNA was further analyzed in three independent homozygous lines using inverse PCR, and confirmed the integration of transgene in non-coding region of genomic DNA. Immunoblot results showed that the level of J1-1 proteins, which was not normally accumulated in unripe fruits, accumulated high in transgenic plants but appeared to differ among transgenic lines. Moreover, the expression of jasmonic acid-biosynthetic genes and pathogenesis-related genes were up-regulated in the transgenic lines, which is co-related with the resistance of J1-1 transgenic plants to anthracnose disease. Consequently, the constitutive expression of J1-1 in transgenic pepper plants provided strong resistance to the anthracnose fungus that was associated with highly reduced lesion formation and fungal colonization. These results implied the significance of the antifungal protein, J1-1, as a useful agronomic trait to control fungal disease

A 2014 Nationwide Survey of the Distribution of Soybean Mosaic Virus (SMV), Soybean Yellow Mottle Mosaic Virus (SYMMV) and Soybean Yellow Common Mosaic Virus (SYCMV) Major Viruses in South Korean Soybean Fields, and Changes from 2012 Isolate Prevalence.

In 2014 symptomatic soybean samples were collected throughout Korea, and were tested for the most important soybean viruses found in Korea, namely Soybean mosaic virus (SMV), Soybean yellow common mosaic virus (SYCMV), and Soybean yellow mottle mosaic virus (SYMMV). SYMMV was most commonly detected, followed by SMV. Only a few samples were found to be infected by SYCMV; of these, three samples were positive for double infection of SYMMV and SYCMV. Phylogenetic analysis of HC–Pro of the SMV isolates collected in 2014 from the eight provinces of Korea showed that most isolates were distinct from the most common Korean isolate detected in 2012, but related to other Korean, Chinese and North American isolates. No isolates varying in HC–Pro amino acid residues implicated in efficiency of RNA silencing suppression activity were detected in 2014. Phylogenetic analysis of ORF1 of both 2012 and 2014 SYCMV isolates showed differentiation into three subgroups. However, the geographical distribution of all three viruses in 2014 was essentially the same as observed in 2012. Quantitative real time PCR data also indicated a similar pattern of dual infected viruses occurrence as existed in 2012. Results showed SMV/ SYMMV double infection RNA accumulation was not changed as much as SYMMV/SYCMV double infection. However, between double infection SMV/SYMMV, SYMMV RNA accumulation level rises more than SMV, and SYCMV RNA accumulation level decline a little compare with SYMMV. In summary, the 2014 survey showed that SMV and SYMMV are still the most prevalent soybean viruses in Korea, and all three viruses were still dispersed in the same areas where they were detected in 2012, although with an apparent shift towards SMV Group I isolates compared to 2012. The reason for the shift in SMV isolates across all Korean provinces is not clear, as seed transmission through farmer–saved seed is presumed to be the main source of infection within the crop

Northern blot analysis of unripe fruits from transgenic pepper lines.

<p>Lane 1, green fruit (G) from wild-type (WT) plant as a negative control; lanes 2–4, three T₁ transgenic plants representing homozygous progenies; lanes 5–7, three T₁ transgenic plants representing hemizygous progenies; lane 8, ripe fruit (R) from WT plant as a positive control. ³²P-labeled <i>J1-1</i> was used as a probe, and total RNAs were shown as loading controls in lower panels.</p

Sequence analysis of T-DNA/gDNA junctions in transgenic pepper lines by i-PCR.

<p>ND, not determined.</p>a<p>Restriction enzyme used for gDNA rescue.</p>b<p>Length of rescued gDNA flanking the T-DNA border.</p>c<p>Deleted length at endpoint of the T-DNA.</p

Southern blot analysis of transgenic pepper plants carrying <i>J1-1</i> gene.

<p><b>A,</b> Schematic diagram of the T-DNA representing restriction enzyme sites and primer sites for i-PCR. LB, T-DNA left border repeat; RB, T-DNA right border repeat; <i>HPT1</i>, hygromycin phosphotransferase I; CaMV35S, CaMV 35S promoter; T_NOS, transcriptional terminator of nopaline synthase (<i>NOS</i>); T_35S, CaMV 35S transcriptional terminator. <b>B,</b> Southern blot analysis. gDNA was digested with <i>Hin</i>dIII, and hybridized with ³²P-labeled <i>HPT1</i> probe (left) or rehybridized with the <i>J1-1</i>gene (right). WT, non-transformed wild-type pepper plant. Arrowheads indicate endogenous J1-1 bands.</p

J1-1 recombinant protein shows antifungal activity against <i>C. gloeosporioides</i>.

<p><b>A,</b> Spore germination. <b>B,</b> Appressorium formation. Spore suspensions were amended with 10 µL of the GST/J1-1 recombinant protein or its heated protein to final concentrations of 0.1 mg·mL⁻¹. The protein was heated by incubating at 90°C for 10 min. A minimum of 100 spores were counted per replicate. Each value represents the mean ± SD of three replicates. Means with different letters in each column are significantly different at P<0.05. <b>C,</b> Representative photos of fungi that were treated with 0.1 mg·mL⁻¹ of GST/J1-1 recombinant protein for 48 hours (right). Control was treated with distilled water (left). Arrows indicate appressorium.</p

Expression of JA-biosynthesis related genes (A) and pathogenesis-related genes (B) in transgenic pepper fruits.

<p>Total RNAs were extracted from the unripe fruits of T₂ transgenic pepper lines (J15, J32, and J51). 10 µg of total RNA was separated in a formaldehyde/agarose gel, transferred onto nylon membrane, and hybridized to radiolabeled respective probes. WT (G), non-transgenic unripe fruits as a negative control; WT (R) non-transgenic ripe fruits as a positive control.</p

Expression of the J1-1 in the unripe pepper fruits of transgenic plants.

<p><b>A,</b> Northern blot analysis of the <i>J1-1</i> transcript. Total RNA from each T₂ progeny was hybridized to a radiolabeled <i>J1-1</i> probe. Lane 1, unripe green fruits (G) of non-transformed wild-type (WT) pepper plant as a negative control; lanes 2–5, four T₂ transgenic lines representing homozygous progenies; lane 6, ripe fruits (R) of non-transformed pepper as a positive control. <b>B,</b> Immunoblot analysis of the J1-1 protein. Total soluble proteins from T₂ progenies were subjected to immunoblot analysis with polyclonal anti-J1-1 antibody. Total RNA and β-tubulin were shown as loading controls.</p

Expression of J1-1 is related to fruit ripening and induced by fungal infection in pepper fruits.

<p><b>A,</b> Organ-specific expression of J1-1 protein in leaves, stems, roots, flowers, unripe (UR) and ripe (R) fruits of pepper. β-tubulin was shown as a loading control. An arrowhead indicates the protein band of J1-1. <b>B,</b> Fungal-induced J1-1 accumulation in unripe and ripe pepper fruits infected with <i>C. gloeosporioides</i>. Numbers on the top represent hours after infection (HAI). Immunoblot analysis was performed with total soluble proteins from pepper tissues using polyclonal J1-1 antibody. <b>C,</b> Immunolocalization of J1-1 in unripe (<b>a</b>–<b>c</b>) and ripe (<b>d</b>–<b>f</b>) fruits at 0, 24, and 48 h after inoculation. To localize the protein, transverse sections of pepper fruits were incubated with polyclonal J1-1 antibody that was detected with AEC (3-amino-9-ethylcarbazole) chromogen, shown as red. The arrows indicate fungal spores on the surface of the pepper fruits. Bar  = 50 µm.</p

2015 Nationwide Survey Revealed Barley stripe mosaic virus in Korean Barley Fields

A seed–transmitted virus has consistently caused significant economic damage to barley crops in Korea in recent years, and may be increasing because many farmers save seed for replanting. Because some barley seed is imported, there is the potential for introduction of new seed–transmitted viruses, causing diseases which may spread. Barley cultivation in South Korea is expanding nationwide due to the increasing popularity of health foods, so both production and quality of barley grain is important. Although Barley stripe mosaic virus (BSMV) has been reported previously as a small percentage of all barley viruses present in Korea, increases in imports of barley seed may lead to increased occurrence of this seed–transmitted virus. We therefore investigated the virus status of barley crops around Iksan and Wanju. On several newly cultivated barley farms, we observed areas showing symptoms typical of BSMV, and confirmed BSMV infection in 24% of the samples examined. In order to understand the occurrence and seed transmission of Korean BSMV isolates, we examined sequence variation within the Triple Gene Block proteins, and subcellular localization of two of these proteins. The newly identified Korean BSMV isolates show low sequence variability and high sequence homology to previously reported US isolates. With these results, we expect to confirm distribution of barley viruses and possible emerging viruses, which will serve as base line data to document virus prevention and control measures