Combination of newly developed SNP and InDel markers for genotyping the <Emphasis Type="Italic">Cf-9</Emphasis> locus conferring disease resistance to leaf mold disease in the tomato

The Cf-9 gene in the tomato is known to confer resistance against leaf mold disease caused by Cladosporium fulvum, and a gene-based marker targeted to the Cf-9 allele has been widely used as a crop protection approach. However, we found this marker to be misleading in genotyping. Therefore, we developed new single-nucleotide polymorphism (SNP) and insertion and deletion (InDel) markers targeted to the Cf-9 allele in order to increase genotyping accuracy and facilitate high-throughput screening. The DNA sequences of reported Cf-9, cf-9, Cf-0, and closely related Cf-4 alleles were compared, and two functional and non-synonymous SNPs were found to distinguish the Cf-9 resistance allele from the cf-9, Cf-0, and Cf-4 alleles. An SNP marker including these two SNPs was developed and applied to the genotyping of 33 tomato cultivars by high-resolution melting analysis. Our SNP marker was able to select all three Cf-9 genotypes (resistant, heterozygous, and susceptible alleles). Interestingly, two cultivars were grouped separately from these three genotypes. To further examine this outgroup, we preformed polymerase chain reaction (PCR) on two InDel regions identified by sequence comparison of the Cf-9 and Cf-4 genes. The band patterns revealed that these two cultivars carried Cf-4 rather than Cf-9 alleles and that three cultivars classified in the Cf-9 resistance group actually carried both Cf-9 and Cf-4 genes. To determine whether these genotyping results were consistent with disease resistance phenotypes, we examined the induction of a hypersensitive response by transiently expressing the corresponding effector genes, and found that the results matched perfectly with the genotyping results. These findings indicate that the combination of our SNP and InDel markers allows resistant Cf-9 alleles to be distinguished from cf-9 and Cf-4 alleles, which will be useful for marker-assisted selection of tomato cultivars resistant to C. fulvum.     

Mapping of the <Emphasis Type="Italic">Cf-6</Emphasis> tomato leaf mould resistance locus using SSR markers

The Cf-6 locus of tomato conferring resistance to the Belarus population of the leaf mould causative agent was mapped to the chromosomal region, located 2.2 and 3.4 cM apart from the microsatellite markers, SSR128 and SSR48, respectively. It was demonstrated that the Cf-6 gene, like the Cf-2/Cf-5 cluster, was located on the short arm of tomato chromosome 6. However, Cf-6 differed from these genes concerning phytopathology and molecular characteristics. Based on the Cf-2 gene sequence, a molecular marker, 2-2C, capable of identification of the Cf-6, Cf-2, and Cf-5 loci, was constructed.     

An NB-LRR gene, <Emphasis Type="Italic">TYNBS1</Emphasis>, is responsible for resistance mediated by the <Emphasis Type="Italic">Ty</Emphasis>-<Emphasis Type="Italic">2 Begomovirus</Emphasis> resistance locus of tomato

Key message

An NB-LRR gene, TYNBS1, was isolated from Begomovirus-resistance locus Ty-2. Transgenic plant analysis revealed that TYNBS1 is a functional resistance gene. TYNBS1 is considered to be synonymous with Ty-2.

Abstract

Tomato yellow leaf curl disease caused by Tomato yellow leaf curl virus (TYLCV) is a serious threat to tomato (Solanum lycopersicum L.) production worldwide. A Begomovirus resistance gene, Ty-2, was introduced into cultivated tomato from Solanum habrochaites by interspecific crossing. To identify the Ty-2 gene, we performed genetic analysis. Identification of recombinant line 3701 confirmed the occurrence of a chromosome inversion in the Ty-2 region of the resistant haplotype. Genetic analysis revealed that the Ty-2 gene is linked to an introgression encompassing two markers, SL11_25_54277 and repeat A (approximately 200 kb). Genomic sequences of the upper and lower border of the inversion section of susceptible and resistant haplotypes were determined. Two nucleotide-binding domain and leucine-rich repeat-containing (NB-LRR) genes, TYNBS1 and TYNBS2, were identified around the upper and lower ends of the inversion section, respectively. TYNBS1 strictly co-segregated with TYLCV resistance, whereas TYNBS2 did not. Genetic introduction of genomic fragments containing the TYNBS1 gene into susceptible tomato plants conferred TYLCV resistance. These results demonstrate that TYNBS1 is a functional resistance gene for TYLCV, and is synonymous with the Ty-2 gene.

     

Allele-specific CAPS marker in a <Emphasis Type="Italic">Ve1</Emphasis> homolog of <Emphasis Type="Italic">Capsicum annuum</Emphasis> for improved selection of <Emphasis Type="Italic">Verticillium dahliae</Emphasis> resistance

Verticillium wilt (Verticillium dahliae) is an economically important disease for many high-value crops. The pathogen is difficult to manage due to the long viability of its resting structures, wide host range, and the inability of fungicides to affect the pathogen once in the plant vascular system. In chile pepper (Capsicum annuum), breeding for resistance to Verticillium wilt is especially challenging due to the limited resistance sources. The dominant Ve locus in tomato (Solanum lycopersicum) contains two closely linked and inversely oriented genes, Ve1 and Ve2. Homologs of Ve1 have been characterized in diverse plant species, and interfamily transfer of Ve1 confers race-specific resistance. Queries in the chile pepper WGS database in NCBI with Ve1 and Ve2 sequences identified one open reading frame (ORF) with homology to the tomato Ve genes. Comparison of the candidate CaVe (Capsicum annuum Ve) gene sequences from susceptible and resistant accessions revealed 16 single nucleotide polymorphisms (SNPs) and several haplotypes. A homozygous haplotype was identified for the susceptible accessions and for resistant accessions. We developed a cleaved amplified polymorphic sequence (CAPS) molecular marker within the coding region of CaVe and screened diverse germplasm that has been previously reported as being resistant to Verticillium wilt in other regions. Based on our phenotyping using the New Mexico V. dahliae isolate, the marker could select resistance accessions with 48% accuracy. This molecular marker is a promising tool towards marker-assisted selection for Verticillium wilt resistance and has the potential to improve the efficacy of chile pepper breeding programs, but does not eliminate the need for a bioassay. Furthermore, this work provides a basis for future research in this important pathosystem.     

Requirement of the isocitrate lyase gene <Emphasis Type="Italic">ICL1</Emphasis> for <Emphasis Type="Italic">VPS41</Emphasis>-mediated starvation response in <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis>

Cryptococcus neoformans is a major cause of fungal meningitis in individuals with impaired immunity. Our previous studies have shown that the VPS41 gene plays a critical role in the survival of Cryptococcus neoformans under nitrogen starvation; however, the molecular mechanisms underlying VPS41-mediated starvation response remain to be elucidated. In the present study, we show that, under nitrogen starvation, VPS41 strongly enhanced ICL1 expression in C. neoformans and that overexpression of ICL1 in the vps41 mutant dramatically suppressed its defects in starvation response due to the loss of VPS41 function. Moreover, targeted deletion of ICL1 resulted in a dramatic decline in viability of C. neoformans cells under nitrogen deprivation. Taken together, our data suggest a model in which VPS41 up-regulates ICL1 expression, directly or indirectly, to promote survival of C. neoformans under nitrogen starvation.     

Usefulness of the Non-conventional <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis> Model to Assess <Emphasis Type="Italic">Candida</Emphasis> Virulence

Invasive candidiasis is caused mainly by Candida albicans, but other Candida species have increasing etiologies. These species show different virulence and susceptibility levels to antifungal drugs. The aims of this study were to evaluate the usefulness of the non-conventional model Caenorhabditis elegans to assess the in vivo virulence of seven different Candida species and to compare the virulence in vivo with the in vitro production of proteinases and phospholipases, hemolytic activity and biofilm development capacity. One culture collection strain of each of seven Candida species (C. albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida metapsilosis, Candida orthopsilosis and Candida parapsilosis) was studied. A double mutant C. elegans AU37 strain (glp-4;sek-1) was infected with Candida by ingestion, and the analysis of nematode survival was performed in liquid medium every 24 h until 120 h. Candida establishes a persistent lethal infection in the C. elegans intestinal tract. C. albicans and C. krusei were the most pathogenic species, whereas C. dubliniensis infection showed the lowest mortality. C. albicans was the only species with phospholipase activity, was the greatest producer of aspartyl proteinase and had a higher hemolytic activity. C. albicans and C. krusei caused higher mortality than the rest of the Candida species studied in the C. elegans model of candidiasis.     

<Emphasis Type="Italic">Candida krusei</Emphasis> and <Emphasis Type="Italic">Candida glabrata</Emphasis> reduce the filamentation of <Emphasis Type="Italic">Candida albicans</Emphasis> by downregulating expression of <Emphasis Type="Italic">HWP1</Emphasis> gene

Pathogenicity of Candida albicans is associated with its capacity switch from yeast-like to hyphal growth. The hyphal form is capable to penetrate the epithelial surfaces and to damage the host tissues. Therefore, many investigations have focused on mechanisms that control the morphological transitions of C. albicans. Recently, certain studies have showed that non-albicans Candida species can reduce the capacity of C. albicans to form biofilms and to develop candidiasis in animal models. Then, the objective of this study was to evaluate the effects of Candida krusei and Candida glabrata on the morphogenesis of C. albicans. Firstly, the capacity of reference and clinical strains of C. albicans in forming hyphae was tested in vitro. After that, the expression of HWP1 (hyphal wall protein 1) gene was determined by quantitative real-time PCR (polymerase chain reaction) assay. For both reference and clinical strains, a significant inhibition of the hyphae formation was observed when C. albicans was incubated in the presence of C. krusei or C. glabrata compared to the control group composed only by C. albicans. In addition, the culture mixed of C. albicans-C. krusei or C. albicans-C. glabrata reduced significantly the expression of HWP1 gene of C. albicans in relation to single cultures of this specie. In both filamentation and gene expression assays, C. krusei showed the higher inhibitory activity on the morphogenesis of C. albicans compared to C. glabrata. C. krusei and C. glabrata are capable to reduce the filamentation of C. albicans and consequently decrease the expression of the HWP1 gene.     

Down-regulation of <Emphasis Type="Italic">Arabidopsis</Emphasis><Emphasis Type="Italic">DND1</Emphasis> orthologs in potato and tomato leads to broad-spectrum resistance to late blight and powdery mildew

Multiple susceptibility genes (S), identified in Arabidopsis, have been shown to be functionally conserved in crop plants. Mutations in these S genes result in resistance to different pathogens, opening a new way to achieve plant disease resistance. The aim of this study was to investigate the role of Defense No Death 1 (DND1) in susceptibility of tomato and potato to late blight (Phytophthora infestans). In Arabidopsis, the dnd1 mutant has broad-spectrum resistance against several fungal, bacterial, and viral pathogens. However this mutation is also associated with a dwarfed phenotype. Using an RNAi approach, we silenced AtDND1 orthologs in potato and tomato. Our results showed that silencing of the DND1 ortholog in both crops resulted in resistance to the pathogenic oomycete P. infestans and to two powdery mildew species, Oidium neolycopersici and Golovinomyces orontii. The resistance to P. infestans in potato was effective to four different isolates although the level of resistance (complete or partial) was dependent on the aggressiveness of the isolate. In tomato, DND1-silenced plants showed a severe dwarf phenotype and autonecrosis, whereas DND1-silenced potato plants were not dwarfed and showed a less pronounced autonecrosis. Our results indicate that S gene function of DND1 is conserved in tomato and potato. We discuss the possibilities of using RNAi silencing or loss-of-function mutations of DND1 orthologs, as well as additional S gene orthologs from Arabidopsis, to breed for resistance to pathogens in crop plants.     

Molecular characterization and expression analysis of the <Emphasis Type="Italic">SPL</Emphasis> gene family with <Emphasis Type="Italic">BpSPL9</Emphasis> transgenic lines found to confer tolerance to abiotic stress in <Emphasis Type="Italic">Betula platyphylla</Emphasis> Suk.

Christolea crassifolia HARDY: gene (CcHRD) belongs to the AP2/ERF-like tanscritpion factor family, and overexpression of HRD gene has been proved to result in improved water use efficiency and enhanced drought resistance in multiple plant species. In the present study, we cloned the CcHRD gene from Christolea crassifolia, which shares 99.1% sequence similarity with the HRD gene from Arabidopsis thaliana. We generated transgenic tomato plants expressing CcHRD gene by agrobacterium-mediated genetic transformation. Our results revealed that the transgenic tomato plants showed a more developed root system and higher fruit yield than the wild-type plants. Furthermore, the leaf relative water content, chlorophyll content and Fv/Fm value in transgenic plants were significantly higher than the wild type, while the relative conductivity and MDA content of transgenic plant leaves were markedly lower than those of wild type under drought stress. We also observed that the major agronomic traits of transgenic tomato plants were improved under natural drought stress compared with those of the wild type. In summary, results in this transgenic study showed that the CcHRD gene could enhance the drought resistance in tomato, and also provided important information for the application of drought-responsive genes in improving crop plant resistance to abiotic stresses.     

<Emphasis Type="Italic">Cantharellus</Emphasis> sect. <Emphasis Type="Italic">Amethystini</Emphasis> in Asia

In this contribution on the genus Cantharellus in Asia, C. subvaginatus is described from the Republic of Korea as a close relative to the Chinese C. vaginatus, which is here reported for the first time from India. Both species are here placed in Cantharellus subg. Cantharellus sect. Amethystini, together with the Indian C. pseudoformosus (syn.: C. umbonatus) and the Malayan C. subamethysteus. As such, Asia has suddenly become the continent with the highest diversity for Amethystini. Species delimitation in sect. Amethystini is molecularly supported by a combined phylogenetic analysis of rDNA sequences obtained for LSU and ITS and additionally suggests the existence of a still undescribed species in North America. Character variability is discussed for all known members of Amethystini, including atypical specimens of the North American C. lewisii that are morphologically more reminiscent of the South Korean C. subvaginatus.