BpWOX11 promotes adventitious root formation in Betula pendula.

Adventitious root formation is a key step in vegetative propagation via cuttings. It is crucial for establishing birch plantations and preserve birch varieties. Although previous studies have highlighted role of WOX11 in controlling adventitious root formation, no such study has been conducted in birch. Understanding the mechanism of adventitious root formation is essential for improvement of rooting or survival rate using stem cuttings in birch. In this study, we cloned BpWOX11 and produced BpWOX11 overexpression (OE) transgenic lines using the Agrobacterium-mediated plant transformation. OE lines exhibited early initiated adventitious root formation, leading to increase the rooting rate of stem cuttings plants. RNA sequencing analysis revealed that OE lines induced the gene expression related to expansin and cell division pathway, as well as defense and stress response genes. These may be important factors for the BpWOX11 gene to promote adventitious root formation in birch cuttings. The results of this study will help to further understand the molecular mechanisms controlling the formation of adventitious roots in birch.

One-Stage Detection without Segmentation for Multi-Type Coronary Lesions in Angiography Images Using Deep Learning.

It is rare to use the one-stage model without segmentation for the automatic detection of coronary lesions. This study sequentially enrolled 200 patients with significant stenoses and occlusions of the right coronary and categorized their angiography images into two angle views: The CRA (cranial) view of 98 patients with 2453 images and the LAO (left anterior oblique) view of 176 patients with 3338 images. Randomization was performed at the patient level to the training set and test set using a 7:3 ratio. YOLOv5 was adopted as the key model for direct detection. Four types of lesions were studied: Local Stenosis (LS), Diffuse Stenosis (DS), Bifurcation Stenosis (BS), and Chronic Total Occlusion (CTO). At the image level, the precision, recall, mAP@0.1, and mAP@0.5 predicted by the model were 0.64, 0.68, 0.66, and 0.49 in the CRA view and 0.68, 0.73, 0.70, and 0.56 in the LAO view, respectively. At the patient level, the precision, recall, and F1scores predicted by the model were 0.52, 0.91, and 0.65 in the CRA view and 0.50, 0.94, and 0.64 in the LAO view, respectively. YOLOv5 performed the best for lesions of CTO and LS at both the image level and the patient level. In conclusion, the one-stage model without segmentation as YOLOv5 is feasible to be used in automatic coronary lesion detection, with the most suitable types of lesions as LS and CTO.

Functional study of BpCOI1 reveals its role in affecting disease resistance in birch.

Plants interact with biotic and abiotic environments. Some of these interactions are detrimental including herbivory consumption and infections by microbial pathogens. The COI1 (coronatine insensitive 1) protein is the master controller of JA-regulated plant responses and plays a regulatory role in the plant defense response. However, there is little information on COI1 function in birch (Betula platyphylla × Betula pendula). Herein, we studied the F-box protein BpCOI1 which is located in the nucleus. To validate the function of this protein, we developed transgenic birch plants with overexpression or repression of BpCOI1 gene. Growth traits, such as tree height, ground diameter, number of lateral branches, did not change significantly among transgenic lines. Alternaria alternata treatment experiments indicated that low expression of BpCOI1 reduced disease resistance in birch. Furthermore, our results showed that low expression of BpCOI1 significantly reduced the sensitivity of plants to exogenous MeJA. Co-expression analysis showed gene expression patterns with similar characteristics. These genes may be closely related in function, or members involved in the same signaling pathway or physiological process with BpCOI 1. The results of transcriptome sequencing and co-expression analysis showed that BpCOI1 affects plant defense against Alternaria alternata by regulating jasmonates. This study reveals the role of BpCOI1 in disease resistance and proposes the possibility of controlling diseases through molecular breeding in birch.

Heritable epigenetic modification of BpPIN1 is associated with leaf shapes in Betula pendula.

The new variety Betula pendula 'Dalecarlica', selected from Betula pendula, shows high ornamental value owing to its lobed leaf shape. In this study, to identify the genetic components of leaf shape formation, we performed bulked segregant analysis and molecular marker-based fine mapping to identify the causal gene responsible for lobed leaves in B. pendula 'Dalecarlica'. The most significant variations associated with leaf shape were identified within the gene BpPIN1 encoding a member of the PIN-FORMED family, responsible for the auxin efflux carrier. We further confirmed the hypomethylation at the promoter region promoting the expression level of BpPIN1, which causes stronger and longer veins and lobed leaf shape in B. pendula 'Dalecarlica'. These results indicated that DNA methylation at the BpPIN1 promoter region is associated with leaf shapes in B. pendula. Our findings revealed an epigenetic mechanism of BpPIN1 in the regulation of leaf shape in Betula  Linn. (birch), which could help in the molecular breeding of ornamental traits.

Construction of a high density genetic linkage map to define the locus conferring seedlessness from Mukaku Kishu mandarin.

Mukaku Kishu ('MK'), a small sized mandarin, is an important source of seedlessness in citrus breeding. Identification and mapping the gene(s) governing 'MK' seedlessness will expedite seedless cultivar development. In this study, two 'MK'-derived mapping populations- LB8-9 Sugar Belle® ('SB') × 'MK' (N=97) and Daisy ('D') × 'MK' (N=68) were genotyped using an Axiom_Citrus56 Array encompassing 58,433 SNP probe sets, and population specific male and female parent linkage maps were constructed. The parental maps of each population were integrated to produce sub-composite maps, which were further merged to develop a consensus linkage map. All the parental maps (except 'MK_D') had nine major linkage groups, and contained 930 ('SB'), 810 ('MK_SB'), 776 ('D') and 707 ('MK_D') SNPs. The linkage maps displayed 96.9 ('MK_D') to 98.5% ('SB') chromosomal synteny with the reference Clementine genome. The consensus map was comprised of 2588 markers including a phenotypic seedless (Fs)-locus and spanned a genetic distance of 1406.84 cM, with an average marker distance of 0.54 cM, which is substantially lower than the reference Clementine map. For the phenotypic Fs-locus, the distribution of seedy and seedless progenies in both 'SB' × 'MK' (55:42, χ2 = 1.74) and 'D' × 'MK' populations (33:35, χ2 = 0.06) followed a test cross pattern. The Fs-locus mapped on chromosome 5 with SNP marker 'AX-160417325' at 7.4 cM in 'MK_SB' map and between two SNP markers 'AX-160536283' and 'AX-160906995' at a distance of 2.4 and 4.9 cM, respectively in 'MK_D' map. The SNPs 'AX-160417325' and 'AX-160536283' correctly predicted seedlessness of 25-91.9% progenies in this study. Based on the alignment of flanking SNP markers to the Clementine reference genome, the candidate gene for seedlessness hovered in a ~ 6.0 Mb region between 3.97 Mb (AX-160906995) to 10.00 Mb (AX-160536283). This region has 131 genes of which 13 genes (belonging to seven gene families) reportedly express in seed coat or developing embryo. The findings of the study will prove helpful in directing future research for fine mapping this region and eventually underpinning the exact causative gene governing seedlessness in 'MK'.

Role of long non-coding RNA in regulatory network response to Candidatus Liberibacter asiaticus in citrus.

Long non-coding RNAs (lncRNAs) serve as crucial regulators in plant response to various diseases, while none have been systematically identified and characterized in response to citrus Huanglongbing (HLB) caused by Candidatus Liberibacter asiaticus (CLas) bacteria. Here, we comprehensively investigated the transcriptional and regulatory dynamics of the lncRNAs in response to CLas. Samples were collected from leaf midribs of CLas- and mock-inoculated HLB-tolerant rough lemon (Citrus jambhiri) and HLB-sensitive sweet orange (C. sinensis) at week 0, 7, 17, and 34 following inoculation using CLas+ budwood of three biological replicates in the greenhouse. A total of 8,742 lncRNAs, including 2,529 novel lncRNAs, were identified from RNA-seq data with rRNA-removed from strand-specific libraries. Genomic variation analyses of conserved lncRNAs from 38 citrus accessions showed that 26 single nucleotide polymorphisms (SNPs) were significantly correlated with HLB. In addition, lncRNA-mRNA weighted gene co-expression network analysis (WGCNA) showed a significant module correlated with CLas-inoculation in rough lemon. Notably, the most significant LNC_28805 and multiple co-expressed genes related to plant defense in the module were targeted by miRNA5021, suggesting that LNC28805 might compete with endogenous miR5021 to maintain the homeostasis of immune gene expression levels. Candidate WRKY33 and SYP121 genes targeted by miRNA5021 were identified as two key hub genes interacting with bacteria pathogen response genes based on the prediction of protein-protein interaction (PPI) network. These two genes were also found within HLB-associated QTL in linkage group 6. Overall, our findings provide a reference for a better understanding of the role of lncRNAs involved in citrus HLB regulation.

Genome-wide identification of the TIFY family reveals JAZ subfamily function in response to hormone treatment in Betula platyphylla.

BACKGROUND: The TIFY family is a plant-specific gene family and plays an important role in plant growth and development. But few reports have been reported on the phylogenetic analysis and gene expression profiling of TIFY family genes in birch (Betula platyphylla). RESULTS: In this study, we characterized TIFY family and identified 12 TIFY genes and using phylogeny and chromosome mapping analysis in birch. TIFY family members were divided into JAZ, ZML, PPD and TIFY subfamilies. Phylogenetic analysis revealed that 12 TIFY genes were clustered into six evolutionary branches. The chromosome distribution showed that 12 TIFY genes were unevenly distributed on 5 chromosomes. Some TIFY family members were derived from gene duplication in birch. We found that six JAZ genes from JAZ subfamily played essential roles in response to Methyl jasmonate (MeJA), the JAZ genes were correlated with COI1 under MeJA. Co-expression and GO enrichment analysis further revealed that JAZ genes were related to hormone. JAZ proteins involved in the ABA and SA pathways. Subcellular localization experiments confirmed that the JAZ proteins were localized in the nucleus. Yeast two-hybrid assay showed that the JAZ proteins may form homologous or heterodimers to regulate hormones. CONCLUSION: Our results provided novel insights into biological function of TIFY family and JAZ subfamily in birch. It provides the theoretical reference for in-depth analysis of plant hormone and molecular breeding design for resistance.

A chromosome-level phased genome enabling allele-level studies in sweet orange: a case study on citrus Huanglongbing tolerance.

Sweet orange originated from the introgressive hybridizations of pummelo and mandarin resulting in a highly heterozygous genome. How alleles from the two species cooperate in shaping sweet orange phenotypes under distinct circumstances is unknown. Here, we assembled a chromosome-level phased diploid Valencia sweet orange (DVS) genome with over 99.999% base accuracy and 99.2% gene annotation BUSCO completeness. DVS enables allele-level studies for sweet orange and other hybrids between pummelo and mandarin. We first configured an allele-aware transcriptomic profiling pipeline and applied it to 740 sweet orange transcriptomes. On average, 32.5% of genes have a significantly biased allelic expression in the transcriptomes. Different cultivars, transgenic lineages, tissues, development stages, and disease status all impacted allelic expressions and resulted in diversified allelic expression patterns in sweet orange, but particularly citrus Huanglongbing (HLB) shifted the allelic expression of hundreds of genes in leaves and calyx abscission zones. In addition, we detected allelic structural mutations in an HLB-tolerant mutant (T19) and a more sensitive mutant (T78) through long-read sequencing. The irradiation-induced structural mutations mostly involved double-strand breaks, while most spontaneous structural mutations were transposon insertions. In the mutants, most genes with significant allelic expression ratio alterations (≥1.5-fold) were directly affected by those structural mutations. In T19, alleles located at a translocated segment terminal were upregulated, including CsDnaJ, CsHSP17.4B, and CsCEBPZ. Their upregulation is inferred to keep phloem protein homeostasis under the stress from HLB and enable subsequent stress responses observed in T19. DVS will advance allelic level studies in citrus.

Seasonal Transcriptome Profiling of Susceptible and Tolerant Citrus Cultivars to Citrus Huanglongbing.

Citrus huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most devastating citrus disease worldwide. Most commercial citrus cultivars are susceptible to HLB, with a few more tolerant exceptions such as 'LB8-9' Sugar Belle mandarin. Transcriptomic analyses have been widely used to investigate the potential mechanisms for disease susceptibility, resistance, or tolerance. Previous transcriptomic studies related to HLB mostly focused on single time point data collection. We hypothesize that changes in day length and temperature throughout the seasons have profound effects on citrus-CLas interactions. Here, we conducted RNA-seq analyses on HLB-susceptible Valencia sweet orange and HLB-tolerant mandarin 'LB8-9' in winter, spring, summer, and fall. Significant variations in differentially expressed genes (DEGs) related to HLB were observed among the four seasons. For both cultivars, the highest number of DEGs were found in the spring. CLas infection stimulates the expression of immune-related genes such as NBS-LRR, RLK, RLCK, CDPK, MAPK pathway, reactive oxygen species (ROS), and PR genes in both cultivars, consistent with the model that HLB is a pathogen-triggered immune disease. HLB-positive mandarin 'LB8-9' trees contained higher concentrations of maltose and sucrose, which are known to scavenge ROS. In addition, mandarin 'LB8-9' showed higher expression of genes involved in phloem regeneration, which might contribute to its HLB tolerance. This study shed light on the pathogenicity mechanism of the HLB pathosystem and the tolerance mechanism against HLB, providing valuable insights into HLB management.

Molecular mechanism of leaf adaxial upward curling caused by BpPIN3 suppression in Betula pendula.

Leaves are one of the vegetative organs of plants that are essential for plant growth and development. PIN-FORMED (PINs) gene is an indoleacetic acid (IAA) transporter that plays a critical role in leaf development. To determine the function of BpPIN3 in leaf polarity formation in Betula pendula, the transgenic lines with BpPIN3 overexpression (OE) and BpPIN3-reduced expression (RE) were analyzed using the Agrobacterium-mediated method. The RE lines displayed the characteristics of leaf margin adaxial upward curling, with lower expression of BpPIN3 resulting in greater rolling. Tissue localization of IAA in the auxin GUS reporter system proved that auxin in the RE was mainly distributed in the secondary veins, palisade tissues, and epidermal cells in the leaf margin area. The auxin content in the leaf margin area was significantly greater than that in the main vein tissue. The cell density of the palisade tissue and the ratio of palisade tissue to spongy tissue in the curled leaf margin of the RE lines were found to be significantly decreased. RNA-seq analysis revealed that the RE hormone-signaling pathway genes were significantly enriched compared with those of the OE and WT lines; in particular, the auxin response-related genes SAURs (i.e., SAUR23, SAUR24, SAUR28, and SAUR50) and GH3.10 were found to be significantly upregulated. qRT-PCR analysis indicated that BpPIN3 expression at the leaf margin was significantly lower than that near the main vein in the RE lines. In contrast, the expression levels of SAURs and GH3.10 were significantly higher than those near the midrib. In conclusion, BpPIN3 regulates the expression of auxin response-related genes and the polar transport of auxin to change the polar form of the proximal and distal axes of birch leaves.

Impact of PaGLK transgenic poplar on microbial community and soil enzyme activity in rhizosphere soil.

Rhizosphere microorganisms are essential parts in maintaining soil ecological functions. Reforestation using genetically modified trees might have great potential to enhance tree production in biotic and abiotic stress, however, their long-term impact on rhizosphere microorganisms is scant. In this study, we studied soil enzyme activities and composition of rhizosphere microorganisms in 2-year-old transgenic PaGLK overexpression (OE), repressed expression (RE) and wild-type (WT) poplar (P. alba × P.berlinensis). The root exudates of PaGLK transgenic poplar (P.alba × P. berlinensis) were analyzed by liquid chromatography-mass spectrometry (LC-MS). The results showed that there were significant difference for soil sucrase, urease, catalase, neutral protease and cellulase between the transgenic and WT lines at different growth periods. Alpha diversity analysis showed that bacterial community abundance and diversity for RE lines were significantly lower than WT (p < 0.05), while RE lines for fungi were significantly higher than WT lines. At the genus level, Burkholderia was the dominant group of rhizosphere bacterial community, and the relative abundance for RE was significantly higher than WT. Tomentella was the dominant group for fungi community. Serendipita for RE was significantly higher than WT and OE. Main metabolite contents of (S)-ACPA, geniposidic acid, agnuside, hydroquinone and pyranocoumarins were significantly different among transgenic lines. These results suggest that transgenic activities have effects on root exudates, rhizosphere soil enzyme activities and soil microbial community composition, but long term effects need to be further investigated.

Role of PsnWRKY70 in Regulatory Network Response to Infection with Alternaria alternata (Fr.) Keissl in Populus.

WRKY is an important complex family of transcription factors involved in plant immune responses. Among them, WRKY70 plays an important role in the process of the plant defense response to the invasion of pathogens. However, the defense mechanism of PsnWRKY70 is not clear in Populus nigra. In this study, we showed that PsnWRKY70-overexpression lines (OE) had fewer leaf blight symptoms than PsnWRKY70-repressing lines (RE). PsnWRKY70 activated MAP kinase cascade genes (PsnM2K4, PsnMPK3, PsnM3K18), calcium channel proteins-related genes (PsnCNG3, PsnCNGC1, PsnCNG4), and calcium-dependent protein kinases genes (PsnCDPKL, PsnCDPKW, PsnCDPKS, PsnCDPKQ). Furthermore, 129 genes of PsnWRKY70 putative genome-wide direct targets (DTGs) were identified by using transcriptome (RNA-seq) and DNA affinity purification sequencing (DAP-seq). PsnWRKY70 directly binds to the promoters of homologous genes and LRR domain proteins to promote the expression of WRKY6, WRKY18, WRKY22, and WRKY22-1, LRR domain proteins LRR8, LRR-RLK, ADR1-like 2, NB-ARC, etc. Our study suggests that PsnWRKY70 enhances the resistance of A. alternata in poplar by activating genes in both pathogen-associated molecular pattern-triggered immunity (PTI) and effector-triggered immunity (ETI).

BpEIN3.1 represses leaf senescence by inhibiting synthesis of ethylene and abscisic acid in Betula platyphylla.

Leaf senescence and abscission play crucial role in annual plant adapting to seasonal alteration and climate changes by shortening life cycle and development process in response to abiotic and/or biotic stressors underlying phytohormones and environmental signals. Ethylene and abscisic acid are the major phytohormones that promotes leaf senescence, involving various transcription factors, such as EIN3 (ethylene-insensitive 3) and EIL (ethylene-insensitive 3-like) gene family, controlling leaf senescence through metabolite biosynthesis and signal transduction pathways. However, the roles of EIN3 regulating leaf senescence responding to environmental changes in perennial plant, especially forestry tree, remain unclear. In this study, we found that BpEIN3.1 from a subordinated to EIL3 subclade, is a transcription repressor and regulated light-dependent premature leaf senescence in birch (Betula platyphylla). BpEIN3.1 might inhibits the transcription of BpATPS1 by binding to its promoter. Shading suppressed premature leaf senescence in birch ein3.1 mutant line. Ethylene and abscisic acid biosynthesis were also reduced. In addition, abscisic acid positively regulated the expression of BpEIN3.1. This was demonstrated by the hormone-response element analysis of BpEIN3.1 promoter and its gene expression after the hormone treatments. Moreover, our results showed that abscisic acid is also involved in maintaining homeostasis. The molecular mechanism of leaf senescence provides a possibility to increasing wood production by delaying of leaf senescence.

Genome-Wide Association Study of Healthful Flavonoids among Diverse Mandarin Accessions.

Mandarins have many unique flavonoids with documented health benefits and that help to prevent chronic human diseases. Flavonoids are difficult to measure and cannot be phenotyped without the use of specialized equipment; consequently, citrus breeders have not used flavonoid contents as selection criteria to develop cultivars with increased benefits for human health or increased tolerance to diseases. In this study, peel, pulp, and seed samples collected from many mandarin accessions and their hybrids were analyzed for the presence of selected flavonoids with documented human health benefits. A genome-wide association study (GWAS) was used to identify SNPs associated with biosynthesis of flavonoids in these mandarin accessions, and there were 420 significant SNPs were found to be associated with 28 compounds in peel, pulp, or seed samples. Four candidate genes involved in flavonoid biosynthesis were identified by enrichment analysis. SNPs that were found to be associated with compounds in pulp samples have the potential to be used as markers to select mandarins with improved phytonutrient content to benefit human health. Mandarin cultivars bred with increased flavonoid content may provide value to growers and consumers.

Phenotypic and Genetic Variation in Morphophysiological Traits in Huanglongbing-Affected Mandarin Hybrid Populations.

Huanglongbing (HLB) caused by 'Candidatus Liberibacter asiaticus' (CLas) is the most costly disease for the global citrus industry. Currently, no effective tools have been found to control HLB. Most commercial citrus varieties are susceptible to HLB, though some citrus hybrid cultivars have reduced sensitivity to the disease. Citrus breeding populations contain a large diversity of germplasm, with thousands of unique genotypes exhibiting a broad range of phenotypes. Understanding phenotypic variation and genetic inheritance in HLB-affected mandarin hybrid populations are crucial for breeding tolerant citrus varieties. In this study, we assessed 448 diverse mandarin hybrids coming from 30 crosses, and 45 additional accessions. For HLB tolerance, we measured HLB severity visual score and CLas titers by qPCR. We also measured seven morphophysiological traits indirectly related to HLB tolerance with leaf area index (LAI), leaf area (LA), leaf mass per area (LMA), photosystem II parameters (Fv/Fo, Fv/Fm), and photochemical performance index (PIabs). By estimating the genetic variation in five half-sib families, we estimated the heritability of phenotypic traits and found a significant genetic effect on HLB visual score and photosynthesis parameters, which indicates opportunities for the genetic improvement of HLB tolerance. In addition, although it is easy to identify infected trees based on HLB symptomatic leaves, visually phenotyping whole trees can be difficult and inconsistent due to the interpersonal subjectivity of characterization. We investigated their relationships and found that LAI was highly correlated with HLB score, with correlation coefficients of r = 0.70 and r = 0.77 for the whole population and five half-sib families, respectively. Photochemical parameters showed significant correlation with HLB severity and responded differentially with the side of the canopy. Our study suggests that LAI and photochemical parameters could be used as a rapid and cost-effective method to evaluate HLB tolerance and inheritance in citrus breeding programs.

Comparative Leaf Volatile Profiles of Two Contrasting Mandarin Cultivars against Candidatus Liberibacter asiaticus Infection Illustrate Huanglongbing Tolerance Mechanisms.

Huanglongbing (HLB), presumably caused by Candidatus Liberibacter asiaticus (CaLas), is a devastating citrus disease worldwide. While all citrus are affected by HLB, some cultivars display greater tolerance; however, the underlying mechanisms are not fully understood. Here, volatile changes in HLB-tolerant LB8-9 Sugar Belle (SB) and HLB-sensitive Murcott mandarins after CaLas infection were comprehensively compared to determine if specific volatiles are associated with HLB responses and to discern the underlying tolerance mechanisms. These cultivars emitted qualitatively and quantitatively different volatiles in response to HLB induced by artificial graft or natural psyllid inoculation. Increasing amounts of total volatiles and de novo-synthesized new volatiles were two key responses to HLB of both cultivars. Markers potentially associated with HLB and host susceptibility were identified. Terpenoid biosynthetic pathway, green leaf volatile, and thymol metabolic pathways responsive to CaLas infection were dramatically altered. SB mandarin allows simultaneous defense and growth, contributing to its greater HLB tolerance.

Utilization of somatic fusion techniques for the development of HLB tolerant breeding resources employing the Australian finger lime (Citrus australasica).

The Australian finger lime is a unique citrus species that has gained importance due to its unique fruit characteristics and perceived tolerance to Huanglongbing (HLB), an often-fatal disease of citrus trees. In this study, we developed allotetraploid finger lime hybrids and cybrids by utilizing somatic cell fusion techniques to fuse diploid 'OLL8' sweet orange or 'Page' tangelo callus-derived protoplasts with finger lime (FL) mesophyll-derived protoplasts. Six somatic fusions were regenerated from the 'OLL8' + FL fusion, while three putative cybrids were regenerated from the 'Page' + FL fusion. Ploidy levels and nuclear-expressed sequence tag derived simple sequence repeat (EST-SSR) markers confirmed the somatic hybrid production, and mitochondrial DNA primer sets confirmed the cybrid nature. Several trees produced by the somatic fusion remained HLB negative even after 6 years of growth in an HLB-endemic environment. Pathogenesis related (PR) and other genes that are often upregulated in HLB-tolerant trees were also upregulated in our somatic fusions. These newly developed somatic fusions and cybrids could potentially be used as breeding parents to develop the next generation of improved HLB-tolerant rootstocks and scions.

The Mechanism of Citrus Host Defense Response Repression at Early Stages of Infection by Feeding of Diaphorina citri Transmitting Candidatus Liberibacter asiaticus.

Citrus Huanglongbing (HLB) is the most devastating disease of citrus, presumably caused by "Candidatus Liberibacter asiaticus" (CaLas). Although transcriptomic profiling of HLB-affected citrus plants has been studied extensively, the initial steps in pathogenesis have not been fully understood. In this study, RNA sequencing (RNA-seq) was used to compare very early transcriptional changes in the response of Valencia sweet orange (VAL) to CaLas after being fed by the vector, Diaphorina citri (Asian citrus psyllid, or ACP). The results suggest the existence of a delayed defense reaction against the infective vector in VAL, while the attack by the healthy vector prompted immediate and substantial transcriptomic changes that led to the rapid erection of active defenses. Moreover, in the presence of CaLas-infected psyllids, several downregulated differentially expressed genes (DEGs) were identified on the pathways, such as signaling, transcription factor, hormone, defense, and photosynthesis-related pathways at 1 day post-infestation (dpi). Surprisingly, a burst of DEGs (6,055) was detected at 5 dpi, including both upregulated and downregulated DEGs on the defense-related and secondary metabolic pathways, and severely downregulated DEGs on the photosynthesis-related pathways. Very interestingly, a significant number of those downregulated DEGs required ATP binding for the activation of phosphate as substrate; meanwhile, abundant highly upregulated DEGs were detected on the ATP biosynthetic and glycolytic pathways. These findings highlight the energy requirement of CaLas virulence processes. The emerging picture is that CaLas not only employs virulence strategies to subvert the host cell immunity, but the fast-replicating CaLas also actively rewires host cellular metabolic pathways to obtain the necessary energy and molecular building blocks to support virulence and the replication process. Taken together, the very early response of citrus to the CaLas, vectored by infective ACP, was evaluated for the first time, thus allowing the changes in gene expression relating to the primary mechanisms of susceptibility and host-pathogen interactions to be studied, and without the secondary effects caused by the development of complex whole plant symptoms.

Association of T2/S-RNase With Self-Incompatibility of Japanese Citrus Accessions Examined by Transcriptomic, Phylogenetic, and Genetic Approaches.

Several citrus varieties show gametophytic self-incompatibility (GSI), which can contribute to seedless fruit production in several cultivars. This study investigated the genes regulating this trait through RNA-seq performed using styles collected from the flowers of Japanese citrus cultivars 'Hyuganatsu,' 'Tosabuntan,' 'Hassaku,' 'Banpeiyu,' and 'Sweet Spring'. We screened the transcripts of putative T2 RNases, i.e., the protein family including all S-RNases from S-RNase-based GSI plants, and constructed a phylogenetic tree using the screened T2 RNases and S-RNases retrieved from citrus genome databases and a public database. Three major clusters (class I-III) were formed, among which, the class III cluster contained family specific subclusters formed by S-RNase and a citrus-specific cluster monophyletic to the S-RNase clusters. From the citrus class III cluster, six transcripts were consistent with the S haplotypes previously determined in Japanese citrus accessions, sharing characteristics such as isoelectric point, extracellular localization, molecular weight, intron number and position, and tissue-specific expression with S-RNases. One T2 RNase gene in self-incompatible Hyuganatsu was significantly down-regulated in the styles of a self-compatible mutant of Hyuganatsu in RNA-seq and qPCR analyses. In addition, the inheritance pattern of some T2 RNase genes was consistent with the pattern of the S haplotype in the progeny population of Hyuganatsu and Tosabuntan. As all results supported citrus self-incompatibility being based on S-RNase, we believe that six T2 RNase genes were S-RNases. The homology comparison between the six T2 RNases and S-RNases recently reported in Chinese citrus revealed that three out of six T2 RNases were identical to S-RNases from Chinese citrus. Thus, the other three T2 RNases were finally concluded to be novel citrus S-RNases involved in self-incompatibility.

Clinical and Surgical Evaluations of Reoperation After Mechanical Mitral Valve Replacement Due to Different Etiologies.

BACKGROUND: This study aimed to evaluate the clinical and surgical characteristics of patients who required reoperation after mechanical mitral valve replacement (MVR). METHODS: We retrospectively identified 204 consecutive patients who underwent reoperation after mechanical MVR between 2009 and 2018. Patients were categorized according the reason for reoperation (perivalvular leakage, thrombus formation, or pannus formation). The patients' medical and surgical records were studied carefully and the rates of in-hospital complications were calculated. RESULTS: The mean age was 51±12 years and 44% of the patients were male. The reasons for reoperation were perivalvular leakage (117 patients), thrombus formation (35 patients), and pannus formation (52 patients). The most common positions for perivalvular leakage were at the 6-10 o'clock positions (proportions of ≥25% for each hour position). Most patients had an interval of >10 years between the original MVR and reoperation. The most common reoperation procedure was re-do MVR (157 patients), and 155 of these patients underwent concomitant cardiac procedures. There were 10 in-hospital deaths and 32 patients experienced complications. The 10-year survival rate was 82.2 ± 3.9% in general, and the group of lowest rate was patients with PVL (77.5 ± 5.2%). The independent risk factors were "male" (4.62, 95% CI 1.57-13.58, P = 0.005) and "Hb <9g/dL before redo MV operation" (3.45, 95% CI 1.13-10.49, P = 0.029). CONCLUSION: Perivalvular leakage was the most common reason for reoperation after mechanical MVR, with a low survival rate in long term follow-up relatively.