An Improvement of Shotgun Proteomics Analysis by Adding Next-Generation Sequencing Transcriptome Data in Orange

BACKGROUND: Shotgun proteomics data analysis usually relies on database search. Because commonly employed protein sequence databases of most species do not contain sufficient protein information, the application of shotgun proteomics to the research of protein sequence profile remains a big challenge, especially to the species whose genome has not been sequenced yet. METHODOLOGY/PRINCIPAL FINDINGS: In this paper, we present a workflow with integrated database to partly address this problem. First, we downloaded the homologous species database. Next, we identified the transcriptome of the sample, created a protein sequence database based on the transcriptome data, and integtrated it with homologous species database. Lastly, we developed a workflow for identifying peptides simultaneously from shotgun proteomics data. CONCLUSIONS/SIGNIFICANCE: We used datasets from orange leaves samples to demonstrate our workflow. The results showed that the integrated database had great advantage on orange shotgun proteomics data analysis compared to the homologous species database, an 18.5% increase in number of proteins identification

Bioinformatics and Expression Analysis of CPMAX2 in Citrange

Transgenic citrange lines with rolABC genes behave rosette branching and extreme dwarfing. To explore the regulatory mechanism of plant hormones in axillary shoot growth of transgenic citrange, a full length cDNA of CPMAX2 was cloned from citrange [Citrus sinensis (L.) Osb × Poncirus trifoliate (L.) Raf.] by RT-PCR in this study. The expression of CPMAX2 was detected in axillary tender leaves of 3 transgenic citrange lines and the wild type. Additionally, we constructed an over-expression vector CPMAX2-pCAMBIA1301 for further study. The results showed that the cDNA sequence and its putative peptide sequence shared 99.66% and 99.14% of identity with its Citrus sinensis ortholog MAX2. The deduced amino acid sequence contained putatively one F-box and two LRR repeat domains that are highly conserved in MAX2 genes. CPMAX2 was obviously down-expressed in tender leaves of 3 transgenic citrange lines compared with the wild type. The results suggest CPMAX2 play an important role in regulating the rosette shoot growth in transgenic citrange with rolABC genes

Identification of the Transcription Factors RAP2-13 Activating the Expression of <i>CsBAK1</i> in Citrus Defence Response to <i>Xanthomonas citri</i> subsp. <i>citri</i>

Citrus canker is a quarantined disease caused by the bacterial plant pathogen Xanthomonas citri subsp. citri (Xcc), which causes persistent surface damage, leaf and fruit drop, and tree decline in citrus plants. The citrus cultivar Citron C-05 (Citrus medica L.) is a disease-resistant genotype identified after years of screening at the National Center for Citrus Improvement (Changsha), which displays allergic, necrotic, and disease-resistant responses to Xcc. In this study, the BAK1 gene was identified in this cultivar to be a disease resistance gene involved in plant-microbe interaction between citrus and Xcc. Functional investigations of this gene revealed that both CsBAK1 (C. sinensis BAK1) or CmBAK1(C. medica BAK1) could inhibit the growth of Xcc to some extent when transiently expressed in the susceptible ‘Bingtang’ genotype of sweet orange. Critical regions of the CmBAK1 promoter sequence were identified by creating downstream deletions and exposing mutants to Xcc to determine effects on the resistance phenotype; a 426 bp region (−2000~–1574) was identified as a key functional region responsible for eliciting the hypersensitive response in plants. Through screening arrayed Citron C-05 cDNA libraries by yeast one-hybrid assays, a basic APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor of CmRAP2-13 that binds directly to the 426 bp key sequence and activates expression of CmBAK1 was identified. Moreover, transcriptional analysis revealed an obvious increase in transcript levels of CsRAP2-13 in Citron C-05, American citron, and Finger citron. In this study, we present the identification of transcriptional activators that are found to interact with BAK1 proteins in response to Xcc. These results reveal a coordinated regulatory mechanism of RAP2-13, which may be involved in defence responses through the regulation of BAK1

Functional Analysis of RMA3 in Response to <em>Xanthomonas citri</em> subsp. <em>citri</em> Infection in Citron C-05 (<em>Citrus medica</em>)

Citrus bacterial canker disease, caused by Xanthomonas citri subsp. citri (Xcc), poses a significant global threat to the citrus industry. Lateral organ boundaries 1 (Lob1) is confirmed as a citrus susceptibility gene that induces pathogenesis by interaction with the PthA4 effector of Xcc. Citron C-05 (Citrus medica) is a Citrus genotype resistant to Xcc. However, there is little information available on the regulation of Lob1 in resistant genotypes, which is important for the breeding of citrus cultivars resistant to canker disease. This study aimed to identify upstream regulatory factors of Lob1 in Citron C-05 and to investigate its function in disease resistance. ‘Bingtang’ sweet orange (C. sinensis), a susceptible genotype, was utilized as the control. cDNA yeast libraries of Xcc-induced Citron C-05 and ‘Bingtang’ sweet orange were constructed. The capacities of ‘Bingtang’ and Citron C-05 were 1.896 × 107 and 2.154 × 107 CFU, respectively. The inserted fragments ranged from 500 to 2000 bp with a 100% recombination rate. The promoter of Lob1 was segmented into two pieces and the P1 fragment from both genotypes was used to construct a bait yeast (PAbAi-CsLob1-P1; PAbAi-CmLob1-P1). Through library screening with the bait yeast, upstream regulators interacting with the Lob1-P1 promoter were identified and then validated using Y1H and dual-luciferase tests. The expression analysis of the three transcript factors indicated that RMA3 was upregulated by inoculation with Xcc in the resistant Citron C-05, but not in the susceptible sweet orange. The overexpression of CsRMA3 in ‘Bingtang’ sweet orange led to reduced canker symptoms, with a significantly lower pathogen density in the leaves following Xcc inoculation. When CmRMA3 was silenced by virus-induced gene silencing (VIGS) in Citron C-05, typical canker symptoms appeared on the CmRMA3-silenced leaves at 15 days post-inoculation with Xcc. Further expression analyses revealed that the CmRMA3 transcription factor suppressed the expression of Lob1. These results suggest that RMA3 participates in the resistant reaction of Citron C-05 to Xcc infection, and such a response might be in relation to its suppression of the expression of the pathogenic gene Lob1

‘Juxiangyuan’ Seedless Orange: A New Mutant with Male and Female Sterility

Seedless is a highly valued commercial characteristic in the citrus industry, both for fresh consumption and for processed markets. In this study, the ‘Succari Sweet Orange’ (WT) and its seedless mutant ‘Juxiangyuan’ (MT), which originated from a bud mutation, were selected to study the formation of a citrus seedless phenotype. Microscopic analysis of MT’s floral organs, including anther and ovary cross-sections, provides insights into its seedless phenotype compared to the original seeded cultivar. Additionally, pollen features, viability, and in vitro germination were examined to determine the cause of seedlessness. MT exhibited significant developmental deformities in both male and female gametes, with pollen grain analysis indicating a high rate of deformity (41.48%), low viability (9.59%), and minimal in vitro germination (9.56%). Hybridization experiments were conducted to assess male and female sterility and pollen incompatibility. Both WT and MT exhibited parthenocarpic development. Notably, MT fruit produced with an average of 3.51 seeds pollinated to WT, despite severe pollen abortion of MT. MT, however, produced seedless fruit through self-breeding or cross-breeding with other varieties, demonstrating stable female sterility. Despite reduced pollen quantity and viability in the seedless mutant ‘Juxiangyuan’, its seedlessness primarily stems from female sterility. This study contributed to a deeper understanding of seedless formation in ‘Juxiangyuan’ and provided valuable information relevant to its commercial cultivation

A sequential treatment with sodium hypochlorite and a reduced dose of imazalil heated at 50 °C effectively control decay of individually film-wrapped lemons stored at 20 °C

Storage of individual seal-packaged citrus fruit at room temperature in China is a very common practice that requires a pre-storage treatment with a high concentration of an effective fungicide as imazalil (IMZ) to prevent decay. In this study, lemons were washed with NaOCl (200 mg L−1) or not, treated with IMZ (50 or 1000 mg L−1) at 20 or 50 °C and individually wrapped with a 16-μm thick extensible polyvinylchloride film (Film A) or two heat shrinkable polyolefinic films, thick 15 (Film B) or 19-μm (Film C). The sequential treatment with NaOCl and IMZ at 50 mg L−1 at 50 °C, was as effective as IMZ at 1000 mg L−1 at 20 °C in controlling Penicillium decay. Losses for decay in fruit wrapped with the two more permeable films (Film A and Film B) never exceeded 10%, while in those wrapped with Film C (the least permeable) peaked to 41%. All films reduced weight losses, which at the end of storage were 11% in fruit wrapped with Film A and below 4% in those wrapped with the other two films, while were 41% in unwrapped ones. After one week of storage, only 50% of unwrapped fruit were marketable whereas all wrapped fruit were still marketable after 8 weeks. Respiration as well as juice acetaldehyde and ethanol were slightly affected by the two more permeable films, while an abnormal production of CO2, acetaldehyde and ethanol occurred in those wrapped with film C. Changes in chemical parameters were relevant in fruit sealed with Film C and minor in those with Film A and Film B. Decay control and quality preservation of lemons stored at room temperature can be achieved for several weeks by a sequential treatment with NaOCl and a heated water emulsion of IMZ at 50 mg L−1, when fruit are wrapped with plastic films highly permeable to gases

Genetic Diversity Analysis of Guangxi Kumquat (<i>Fortunella</i> Swing) Germplasm Using SRAP Markers

In order to understand the genetic diversity of germplasm resources of kumquats in Guangxi, 14 kumquat germplasm resources in Guangxi and 12 accessions from other provinces were analyzed by using SRAP markers. In total, 19 primer pairs with high stability, good reproducibility, and high polymorphism were chosen for analysis of all 26 kumquat genotypes. Among the 104 amplified bands, 90 (86.54%) were polymorphic. SRAP markers were analyzed by employing Principal Coordinate Analysis, Population Structure Analysis, and Hierarchical Cluster Analysis (UPGMA). The classification results showed that the 26 kumquat germplasm resources could be divided into 5 groups, including cultivated kumquat, intergeneric hybrid, wild kumquat from other provinces, wild kumquat, and hybrid kumquat from Guangxi. The Guangxi kumquat germplasm had high genetic diversity, and were clearly divided into three groups: cultivated kumquat, wild kumquat, and hybrid kumquat. Additionally, the eight cultivated kumquat varieties in Guangxi were further divided into two subgroups. Wild kumquat in Guangxi or in other provinces belonged to different groups; meanwhile, the Guangxi kumquat hybrid formed an independent group, thus indicating that Guangxi wild kumquat and hybrid kumquat possess certain specificity, or they possibly belong to different species. Among the tested 26 kumquat accessions, 23 unique genotype-specific SRAP markers were detected for 14 kumquat genotypes, which were positively identified. For the remaining 12 accessions without genotype-specific markers, they were distinguished by various combinations of markers. These results may have certain importance for kumquat genetic research and cultivar selection

High throughput sequencing of a stem pitting citrus tristeza virus isolate from Hunan Province P.R. China

A stem-pitting isolate of citrus tristeza virus (CTV), spreading in Hunan province of &nbsp;China (HU-PSTS), was sequenced and indexed on indicator plants. Biological assays showed that HU-PSTS is a highly aggressive stem pitting isolate belonging to biogroup 5. Viral small RNAs (18-26 nt) of the isolate were deep sequenced by Illumina technology to gain genomic information on the CTV strain infecting the source plant. The reads mapping with 17 CTV reference genomes enabled us to re-assemble the genomes of VT, T68, T30 and T3 strains. Among the VT, the highest number of mapped reads (47-41%) was with SG29, T318A, CT11A, Nuaga and AT-1 genomes, whereas T68, T30 and T3 genomes were less represented (28-20%). Alignments with genomes belonging to T36 and RB strains revealed percentage of mapped reads ranging from 10 to 12%. This is the first sequenced genome of a CTV isolate from Hunan province. According to the results, further sequencing and bioindexing need to be developed to explore the potential of a local cross protective strategy