Joung-Ho Lee

Aplasia of the internal carotid artery.

Summary. Background: The majority of previous reports on this rare agenesis of the internal carotid artery (ICA) have been limited to reporting upon its association with other congenital anomalies case by case. In order to collectively summarize this congenital anomaly of ICA, we have reviewed nine cases of ICA aplasia and their associated abnormalities. Method: Nine cases of ICA aplasia were reviewed. The diagnosis of aplasia or agenesis of the ICA was based on angiographic findings and the presence of an absent or hypoplastic bony carotid canal by temporal bone computed tomography (TBCT). Their presumable embryological aetiologies, initial presenting symptoms, unusual collateral circulations, as demonstrated by angiographies, and various associated anomalies are reviewed. Findings: The initial presentations were; subarachnoid haemorrhage in three patients, headache in one patient and ischemic symptoms and signs in three patients. The remaining two cases were found incidentally during angiography for other diseases. Collateral circulations to the middle cerebral artery ipsilateral to the ICA aplasia were via posterior communicating artery (P-com) or anterior communicating artery (A-com). On TBCT, all cases but one demonstrated agenesis of the bony carotid canal and the remaining case showed a hypoplastic canal. Cerebral aneurysms were found in six patients, four with A-com aneurysm, one with a basilar bifurcation aneurysm, and one with both a right P-com and a left cavernous ICA aneurysm; two incidentally found cases had no aneurysm. Other associated abnormalities were found in four cases; one case of hypoplasia of the common carotid artery (CCA) with an arachnoid cyst at the temporal pole, one case of abnormal origin of the right CCA from the aorta and the right subclavian artery from the descending aorta, one case of congenital temporomandibular joint (TMJ) ankylosis, and one case of nasopharyngeal angiofibroma with atresia of the upper basilar artery. Except for the atresia of the upper basilar artery, all such abnormalities were found on the same side as the ICA aplasia. Interpretation: Agenesis or aplasia of ICA may be entirely harmless. However, associated conditions such as cerebral aneurysm or abnormal collateral channels should alert clinicians to the possibility of deterioration to life-threatening conditions, such as subarachnoid haemorrhage or irreversible ischemia. Other associated anomalies are commonly depicted on the same side as the ICA aplasia and may also give rise to issues of clinical importance.

Plant Translation Elongation Factor 1Bβ Facilitates Potato Virus X (PVX) Infection and Interacts with PVX Triple Gene Block Protein 1

The eukaryotic translation elongation factor 1 (eEF1) has two components: the G-protein eEF1A and the nucleotide exchange factor eEF1B. In plants, eEF1B is itself composed of a structural protein (eEF1Bγ) and two nucleotide exchange subunits (eEF1Bα and eEF1Bβ). To test the effects of elongation factors on virus infection, we isolated eEF1A and eEF1B genes from pepper (Capsicum annuum) and suppressed their homologs in Nicotiana benthamiana using virus-induced gene silencing (VIGS). The accumulation of a green fluorescent protein (GFP)-tagged Potato virus X (PVX) was significantly reduced in the eEF1Bβ- or eEF1Bɣ-silenced plants as well as in eEF1A-silenced plants. Yeast two-hybrid and co-immunoprecipitation analyses revealed that eEF1Bα and eEF1Bβ interacted with eEF1A and that eEF1A and eEF1Bβ interacted with triple gene block protein 1 (TGBp1) of PVX. These results suggest that both eEF1A and eEF1Bβ play essential roles in the multiplication of PVX by physically interacting with TGBp1. Furthermore, using eEF1Bβ deletion constructs, we found that both N- (1-64 amino acids) and C-terminal (150-195 amino acids) domains of eEF1Bβ are important for the interaction with PVX TGBp1 and that the C-terminal domain of eEF1Bβ is involved in the interaction with eEF1A. These results suggest that eEF1Bβ could be a potential target for engineering virus-resistant plants.

Isolation and Characterization of Pepper Genes Interacting with the CMV-P1 Helicase Domain

Cucumber mosaic virus (CMV) is a destructive pathogen affecting Capsicum annuum (pepper) production. The pepper Cmr1 gene confers resistance to most CMV strains, but is overcome by CMV-P1 in a process dependent on the CMV-P1 RNA1 helicase domain (P1 helicase). Here, to identify host factors involved in CMV-P1 infection in pepper, a yeast two-hybrid library derived from a C. annuum ‘Bukang’ cDNA library was screened, producing a total of 76 potential clones interacting with the P1 helicase. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 genes putatively involved in virus infection. The candidate host genes were silenced in Nicotiana benthamiana plants that were then inoculated with CMV-P1 tagged with the green fluorescent protein (GFP). Plants silenced for seven of the genes showed development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Silencing formate dehydrogenase and calreticulin-3 precursor led to reduced virus accumulation. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, but not in upper (systemic) leaves. In the calreticulin-3 precursor-silenced plants, infection was not observed in either the inoculated or the upper leaves. Our results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection.

QTL mapping and GWAS reveal candidate genes controlling capsaicinoid content in Capsicum

Summary Capsaicinoids are unique compounds produced only in peppers (Capsicum spp.). Several studies using classical quantitative trait loci (QTLs) mapping and genomewide association studies (GWAS) have identified QTLs controlling capsaicinoid content in peppers; however, neither the QTLs common to each population nor the candidate genes underlying them have been identified due to the limitations of each approach used. Here, we performed QTL mapping and GWAS for capsaicinoid content in peppers using two recombinant inbred line (RIL) populations and one GWAS population. Whole‐genome resequencing and genotyping by sequencing (GBS) were used to construct high‐density single nucleotide polymorphism (SNP) maps. Five QTL regions on chromosomes 1, 2, 3, 4 and 10 were commonly identified in both RIL populations over multiple locations and years. Furthermore, a total of 109 610 SNPs derived from two GBS libraries were used to analyse the GWAS population consisting of 208 C. annuum‐clade accessions. A total of 69 QTL regions were identified from the GWAS, 10 of which were co‐located with the QTLs identified from the two biparental populations. Within these regions, we were able to identify five candidate genes known to be involved in capsaicinoid biosynthesis. Our results demonstrate that QTL mapping and GBS‐GWAS represent a powerful combined approach for the identification of loci controlling complex traits.

Identification and molecular genetic mapping of Chili veinal mottle virus (ChiVMV) resistance genes in pepper ( Capsicum annuum )

Chili veinal mottle virus (ChiVMV) threatens the agricultural production of peppers (Capsicum annuum) in Asia and Africa. In this study, we evaluated ChiVMV resistance in the four pepper varieties CV3, CV4, CV8, and CV9. Segregation analyses revealed that CV3 and CV8 contain the single dominant resistance gene Cvr1, and CV9 contains the single recessive resistance gene cvr4. SNP markers were developed and used to map the Cvr1 gene in CV3 to the short arm of chromosome 6 where NLR genes are clustered. In CV4 oligogenic resistance loci were detected. A genotyping-by-sequencing (GBS) combined with modified sliding window approach mapped two resistance loci, to chromosomes 6 and 10. The development of SNP markers and the resulting knowledge of genomic positioning will assist in breeding ChiVMV-resistant pepper varieties and in the fine mapping of ChiVMV resistance genes.

Development of Bi gene-based SNP markers for genotyping for bitter-free cucumber lines

High contents of cucurbitacin compounds result in a bitter taste in cucumbers. The Bi locus mapped to chromosome 6 is responsible for cucumber bitterness. The Bi gene encodes oxidosqualene cyclase, a key enzyme in the cucurbitacin biosynthetic pathway. Two alleles of the Bi gene responsible for the phenotypic variation in bitterness differ by a single point mutation in the coding region. An efficient genotyping system is necessary to allow breeders to screen for bitterness alleles in their breeding programs. In the present study, the single nucleotide polymorphism located within the Bi gene, which is directly linked to cucumber bitterness, was utilized for gene-based marker development. We developed reliable and cost-effective high-resolution melting (HRM)- and Kompetitive Allele-Specific PCR (KASP)-based molecular markers (BiHRM1 and Bi-KASP). These gene-based markers should enhance the accuracy and effectiveness of marker-assisted selection for bitter-free cucumber lines in cucumber breeding programs.

Identification of Cucumber mosaic resistance 2 (cmr2) That Confers Resistance to a New Cucumber mosaic virus Isolate P1 (CMV-P1) in Pepper (Capsicum spp.)

Cucumber mosaic virus (CMV) is one of the most devastating phytopathogens of Capsicum. The single dominant resistance gene, Cucumber mosaic resistant 1 (Cmr1), that confers resistance to the CMV isolate P0 has been overcome by a new isolate (CMV-P1) after being deployed in pepper (Capsicum annuum) breeding for over 20 years. A recently identified Indian C. annuum cultivar, “Lam32,” displays resistance to CMV-P1. In this study, we show that the resistance in “Lam32” is controlled by a single recessive gene, CMV resistance gene 2 (cmr2). We found that cmr2 conferred resistance to CMV strains including CMV-Korean, CMV-Fny, and CMV-P1, indicating that cmr2 provides a broad-spectrum type of resistance. We utilized two molecular mapping approaches to determine the chromosomal location of cmr2. Bulked segregant analysis (BSA) using amplified fragment-length polymorphism (AFLP) (BSA-AFLP) revealed one marker, cmvAFLP, located 16 cM from cmr2. BSA using the Affymetrix pepper array (BSA-Affy) identified a single-nucleotide polymorphism (SNP) marker (Affy4) located 2.3 cM from cmr2 on chromosome 8. We further screened a pepper germplasm collection of 4,197 accessions for additional CMV-P1 resistance sources and found that some accessions contained equivalent levels of resistance to that of “Lam32.” Inheritance and allelism tests demonstrated that all the resistance sources examined contained cmr2. Our result thus provide genetic and molecular evidence that cmr2 is a single recessive gene that confers to pepper an unprecedented resistance to the dangerous new isolate CMV-P1 that had overcome Cmr1.