A rice chitinase cDNA (RCC2) driven by the CaMV 35S promoter was introduced into cucumber (Cucumis sativus L.) through Agrobacterium mediation. More than 200 putative transgenic shoots were regenerated and grown on MS medium supplemented with 100 mg/l kanamycin. Sixty elongated shoots were examined for the presence of the integrated RCC2 gene and subsequently confirmed to have it. Of these, 20 were tested for resistance against gray mold (Botrytis cinerea) by infection with the conidia: 15 strains out of the 20 independent shoots exhibited a higher resistance than the control (non-transgenic plants). Three transgenic cucumber strains (designated CR29, CR32 and CR33) showed the highest resistance against B. cinerea: the spread of disease was inhibited completely in these strains. Chitinase gene expression in highly resistant transgenic strains (CR32 and CR33) was compared to that of a susceptible transgenic strain (CR20) and a control. Different responses for disease resistance were observed among the highly resistant strains. CR33 inhibited appressoria formation and penetration of hyphae. Although CR32 permitted penetration of hyphae, invasion of the infection hyphae was restricted. Furthermore, progenies of CR32 showed a segregation ratio of 3:1 (resistant:susceptible). As the disease resistance against gray mold was confirmed to be inheritable, these highly resistant transgenic cucumber strains would serve as good breeding materials for disease resistance.

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens.

An improved method for genetic transformation of cucumber (Cucumis sativus L. cv. Shinhokusei No. 1) was developed. Vacuum infiltration of cotyledonary explants with Agrobacterium suspension enhanced the efficiency of Agrobacterium infection in the proximal regions of explants. Co-cultivation on filter paper wicks suppressed necrosis of explants, leading to increased regeneration efficiency. Putative transgenic plants were screened by kanamycin resistance and green fluorescent protein (GFP) fluorescence, and integration of the transgene into the cucumber genome was confirmed by genomic polymerase chain reaction (PCR) and Southern blotting. These transgenic plants grew normally and T1 seeds were obtained from 7 lines. Finally, stable integration and transmission of the transgene in T1 generations were confirmed by GFP fluorescence and genomic PCR. The average transgenic efficiency for producing cucumbers with our method was 11.9 ± 3.5 %, which is among the highest values reported until date using kanamycin as a selective agent.Electronic supplementary materialThe online version of this article (doi:10.1007/s11816-012-0260-1) contains supplementary material, which is available to authorized users.

We investigated estrogen-inducible green fluorescent protein (GFP) expression patterns using an estrogen receptor fused chimeric transcription activator, XVE, in the monocotyledonous model plant rice (Oryza sativa L.). This system has been shown to be an effective chemical-inducible gene expression system in Arabidopsis and has been applied to other plants in order to investigate gene functions or produce marker-free transgenic plants. However, limited information is available on the correlation between inducer concentration and the expression level of the gene induced in monocots. Here, we produced a transgenic rice integrated estrogen-inducible GFP expression vector, pLex:GFP, and investigated dose–response and time-course patterns of GFP induction in rice calli and seedlings for the first time. With 17-β-estradiol treatment at >5 μM, GFP signals were detected in the entire surface of calli within 2 days of culture. Highest GFP signals were extended for 8 days with estradiol treatment at 25 μM. In three-leaf-stage seedlings, GFP signals in the leaves of pLex:GFP-integrated transgenic lines were weaker than those in the leaves of p35S:GFP-integrated transgenic lines. However, GFP signals in the roots of pLex:GFP- and p35S:GFP-integrated transgenic lines were similar with estradiol treatment at >10 μM. With regard to controlling appropriate gene expression, these results might provide helpful indications on estradiol treatment conditions to be used for the XVE system in rice and other monocots.

To characterize Japanese encephalitis virus (JEV) strains recently prevalent in Japan, JEV surveillance was performed in pigs from 2002 to 2004. Eleven new JEV isolates were obtained and compared with previous isolates from Japan and other Asian countries. All of the isolates were classified into genotype 1 by nucleotide sequence analysis of the E gene. Two new isolates with different levels of neurovirulence and neuroinvasiveness, but with only one nucleotide difference in the E gene, Sw/Mie/34/2004 and Sw/Mie/40/2004, were isolated at the same farm on the same day. Sw/Mie/40/2004 displayed higher neurovirulence and neuroinvasiveness in mice than the other four new isolates. Another new isolate, Sw/Hiroshima/25/2002, was neutralized by antiserum to Beijing-1 at a level similar to the homologous Beijing-1 strain, whilst seven other new isolates were neutralized at 10-fold-lower titres. However, there were no amino acid differences in the E protein among these eight isolates. The present study indicated that the 11 new JEV isolates were genetically similar, but biologically and serologically heterogeneous. The GenBank/EMBL/DDBJ accession numbers for the E gene and 39 NTR sequences of the 14 JEV isolates determined in this study (-indicates 'data not available'