Bacteriomic Analyses of Asian Citrus Psyllid and Citrus Samples Infected With "Candidatus Liberibacter asiaticus" in Southern California and Huanglongbing Management Implications.

Citrus Huanglongbing (HLB; yellow shoot disease) is associated with an unculturable α-proteobacterium "Candidatus Liberibacter asiaticus" (CLas). HLB was found in southern California in 2012, and the current management strategy is based on suppression of the Asian citrus psyllid (Diaphorina citri) that transmits CLas and removal of confirmed CLas-positive trees. Little is known about Asian citrus psyllid-associated bacteria and citrus-associated bacteria in the HLB system. Such information is important in HLB management, particularly for accurate detection of CLas. Recent advancements in next-generation sequencing technology provide new opportunities to study HLB through genomic DNA sequence analyses (metagenomics). In this study, HLB-related bacteria in Asian citrus psyllid and citrus (represented by leaf midrib tissues) samples from southern California were analyzed. A metagenomic pipeline was developed to serve as a prototype for future bacteriomic research. This pipeline included steps of next-generation sequencing in Illumina platform, de novo assembly of Illumina reads, sequence classification using the Kaiju tool, acquisition of bacterial draft genome sequences, and taxonomic validation and diversity evaluation using average nucleotide identity. The identified bacteria in Asian citrus psyllids and citrus together included Bradyrhizobium, Buchnera, Burkholderia, "Candidatus Profftella armature," "Candidatus Carsonella ruddii," CLas, Mesorhizobium, Paraburkholderia, Pseudomonas, and Wolbachia. The whole genome of a CLas strain recently found in San Bernardino County was sequenced and classified into prophage typing group 1 (PTG-1), one of the five known CLas groups in California. Based on sequence similarity, Bradyrhizobium and Mesorhizobium were identified as possible source that could interfere with CLas detection using the 16S rRNA gene-based PCR commonly used for HLB diagnosis, particularly at low or zero CLas titer situation.

PRD-2 directly regulates casein kinase I and counteracts nonsense-mediated decay in the Neurospora circadian clock.

Circadian clocks in fungi and animals are driven by a functionally conserved transcription-translation feedback loop. In Neurospora crassa, negative feedback is executed by a complex of Frequency (FRQ), FRQ-interacting RNA helicase (FRH), and casein kinase I (CKI), which inhibits the activity of the clock's positive arm, the White Collar Complex (WCC). Here, we show that the prd-2 (period-2) gene, whose mutation is characterized by recessive inheritance of a long 26 hr period phenotype, encodes an RNA-binding protein that stabilizes the ck-1a transcript, resulting in CKI protein levels sufficient for normal rhythmicity. Moreover, by examining the molecular basis for the short circadian period of upf-1prd-6 mutants, we uncovered a strong influence of the Nonsense Mediated Decay pathway on CKI levels. The finding that circadian period defects in two classically derived Neurospora clock mutants each arise from disruption of ck-1a regulation is consistent with circadian period being exquisitely sensitive to levels of casein kinase I.

Myb transcription factors and light regulate sporulation in the oomycete Phytophthora infestans.

Life cycle progression in eukaryotic microbes is often influenced by environment. In the oomycete Phytophthora infestans, which causes late blight on potato and tomato, sporangia have been reported to form mostly at night. By growing P. infestans under different light regimes at constant temperature and humidity, we show that light contributes to the natural pattern of sporulation by delaying sporulation until the following dark period. However, illumination does not permanently block sporulation or strongly affect the total number of sporangia that ultimately form. Based on measurements of sporulation-induced genes such as those encoding protein kinase Pks1 and Myb transcription factors Myb2R1 and Myb2R3, it appears that most spore-associated transcripts start to rise four to eight hours before sporangia appear. Their mRNA levels oscillate with the light/dark cycle and increase with the amount of sporangia. An exception to this pattern of expression is Myb2R4, which is induced several hours before the other genes and declines after cultures start to sporulate. Transformants over-expressing Myb2R4 produce twice the number of sporangia and ten-fold higher levels of Myb2R1 mRNA than wild-type, and chromatin immunoprecipitation showed that Myb2R4 binds the Myb2R1 promoter in vivo. Myb2R4 thus appears to be an early regulator of sporulation. We attempted to silence eight Myb genes by DNA-directed RNAi, but succeeded only with Myb2R3, which resulted in suppressed sporulation. Ectopic expression studies of seven Myb genes revealed that over-expression frequently impaired vegetative growth, and in the case of Myb3R6 interfered with sporangia dormancy. We observed that the degree of silencing induced by a hairpin construct was correlated with its copy number, and ectopic expression was often unstable due to epigenetic silencing and transgene excision.

Oomycete pathogens encode RNA silencing suppressors.

Effectors are essential virulence proteins produced by a broad range of parasites, including viruses, bacteria, fungi, oomycetes, protozoa, insects and nematodes. Upon entry into host cells, pathogen effectors manipulate specific physiological processes or signaling pathways to subvert host immunity. Most effectors, especially those of eukaryotic pathogens, remain functionally uncharacterized. Here, we show that two effectors from the oomycete plant pathogen Phytophthora sojae suppress RNA silencing in plants by inhibiting the biogenesis of small RNAs. Ectopic expression of these Phytophthora suppressors of RNA silencing enhances plant susceptibility to both a virus and Phytophthora, showing that some eukaryotic pathogens have evolved virulence proteins that target host RNA silencing processes to promote infection. These findings identify RNA silencing suppression as a common strategy used by pathogens across kingdoms to cause disease and are consistent with RNA silencing having key roles in host defense.

Myb transcription factors in the oomycete Phytophthora with novel diversified DNA-binding domains and developmental stage-specific expression.

Transcription factors containing two or three imperfect tandem repeats of the Myb DNA-binding domain (named R2R3 and R1R2R3, respectively) regulate important processes in growth and development. This study characterizes the structure, evolution, and expression of these proteins in the potato pathogen Phytophthora infestans and other oomycetes. P. infestans was found to encode five R2R3 and nine R1R2R3 transcription factor-like proteins, plus several with additional configurations of Myb domains. Sets of R2R3 and R1R2R3 orthologs are well-conserved in three Phytophthora species. Analyses of sites that bind DNA in canonical Myb transcription factors, such as mammalian c-Myb, revealed unusual diversification in the DNA recognition helices of the oomycete proteins. While oomycete R2R3 proteins contain c-Myb-like helices, R1R2R3 proteins exhibit either c-Myb-like or novel sequences. This suggests divergence in their DNA-binding specificities, which was confirmed by electrophoretic mobility shift assays. Eight of the P. infestans R2R3 and R1R2R3 genes are up-regulated during sporulation and three during zoospore release, which suggests their involvement in spore development. This is supported by the observation that an oomycete that does not form zoospores, Hyaloperonospora arabidopsidis, contains one-third fewer of these genes than Phytophthora.

A motif within a complex promoter from the oomycete Phytophthora infestans determines transcription during an intermediate stage of sporulation.

Sporulation in Phytophthora infestans is associated with a major remodeling of the transcriptome. To better understand promoter structure and how sporulation-specific expression is determined in this organism, the Pks1 gene was analyzed. Pks1 encodes a protein kinase that is induced at an intermediate stage of sporulation, prior to sporangium maturation. Major and minor transcription start sites mapped throughout the promoter, which contains many T-rich stretches and Inr-like elements. Within the T-rich region are several motifs which bound nuclear proteins in EMSA. Tests of modified promoters in transformants implicated a CCGTTG located 110-nt upstream of the transcription start point as a major regulator of sporulation-specific transcription. The motif also bound a sporulation-specific nuclear protein complex. A bioinformatics analysis indicated that the motif is highly over-represented within co-expressed promoters, in which it predominantly resides 100-300-nt upstream of transcription start sites. Other sequences, such as a CATTTGTT motif, also bound nuclear proteins but did not play an essential role in spore-specific expression.

Effect of porous glass-ceramic fillers on mechanical properties of light-cured dental resin composites.

OBJECTIVES: The aim of this work was to study the effect of porous particles on the mechanical properties of dental resin composites. METHODS: Two kinds of glass-ceramic powders with different calcium-mica to fluorapatite ratio were used as inorganic fillers for light-cured dental resin composites. The glass-ceramic particles were etched to introduce porous structures. The microstructures of particles and the fractography of dental composites were observed using SEM/EDS. The crystalline phases in the fillers were determined by XRD. The specific surface areas (SBET) of porous fillers were tested to show the degree of porosity. 6 samples were prepared for each bending strength test, which was conducted in a Mechanical Testing System. RESULTS: The results show that the bending strength does not vary monotonically with the porosity of the particles. For the glass-ceramics with a high nominal calcium-mica to fluorapatite ratio, the dental resin filled with porous particles has a higher strength than that filled with dense particles; however, for the glass-ceramics with a low nominal calcium-mica to fluorapatite ratio, the dental resin filled with porous particles has a lower strength. SIGNIFICANCE: The study indicates that the porosity itself is not a decisive factor for strengthening of dental resin composites. The composition of the glass-ceramic particles and the porous structure are also essential factors influencing the mechanical strength. The investigation on the effect of porous glass-ceramic fillers will provide more understanding on the mechanical properties of dental resin composites and will help with the design of new dental restorative materials.

Architecture of the sporulation-specific Cdc14 promoter from the oomycete Phytophthora infestans.

The Cdc14 gene of Phytophthora infestans is transcribed specifically during sporulation, with no mRNA detectable in vegetative hyphae, and is required for sporangium development. To unravel the mechanisms regulating its transcription, mutated Cdc14 promoters plus chimeras of selected Cdc14 sequences and a minimal promoter were tested in stable transformants. This revealed that a tandem repeat of three copies of the motif CTYAAC, located between 67 and 90 nucleotides (nt) upstream of the major transcription start site, is sufficient to determine sporulation-specific expression. All three repeats need to be present for activity, suggesting that they bind a transcription factor through a cooperative mechanism. Electrophoretic mobility shift assays indicated that the CTYAAC repeats are specifically bound by a protein in nuclear extracts. Evidence was also obtained for a second region within the promoter that activates Cdc14 transcription during sporulation which does not involve those repeats. The CTYAAC motif also affects the specificity of transcription initiation. Wild-type Cdc14 is transcribed from a major start site and minor site(s) located about 100 nt upstream of the major site. However, stepwise mutations through the CTYAAC triad caused a graded shift to the upstream sites, as did mutating bases surrounding the major start site; transcripts initiated from the upstream site remained sporulation specific. Replacing the Cdc14 initiation region with the Inr-like region of the constitutive Piexo1 gene had no apparent effect on the pattern of transcription. Therefore, this study reports the first motif determining sporulation-induced transcription in oomycetes and helps define oomycete core promoters.

Preparation of mica-based glass-ceramics with needle-like fluorapatite.

OBJECTIVES: The aim of this study was to prepare mica-based glass-ceramics containing needle-like fluorapatites, and to understand the relationship between the composition, the microstructure and mechanical properties. METHODS: The specimens were prepared by casting and subsequent heat treatment. The crystalline phases in the specimens were determined by X-ray diffraction (XRD). The microstructures of the fractured surface were examined by using a scanning electron microscope with an energy dispersive spectrometer (SEM/EDS). The Vickers hardness and fracture toughness were determined by the indentation method. Statistical analysis of the results was performed by one-way analysis of variance (ANOVA) and Tukey's test. The bioactivity of the glass-ceramics was evaluated by soaking the polished specimens in the simulated body fluid (SBF). RESULTS: Needle-like fluorapatite crystals were successfully obtained in mica-based glass-ceramics by controlling the heat treatment process. The formation of needle-like fluorapatite crystals, instead of particle-like crystals reported in previous studies, can be attributed to the one-dimensional rapid growth of fluorapatite along the c-axis. The mechanical properties of the glass-ceramics are related to the fluorapatite content. The higher the fluorapatite content, the higher the Vickers hardness and the fracture toughness (p<0.01). The bioactivity test showed that the needle-like fluorapatite-containing glass-ceramics possess very good bioactivity. SIGNIFICANCE: As the needle-like fluorapatite crystal has the same morphology as human bones, it endows the mica-based glass-ceramics with good bioactivity. Moreover, the needle-like fluorapatite crystal toughens the base glass-ceramics. Therefore, the mica-based glass-ceramics with needle-like fluorapatite have the potential for the restoration of bone defects and implants.

[Preparation and properties of medical calcium phosphate cement].

The preparation of tetracalcium phosphate (Ca4(PO4)2O, TTCP)was studied. Then calcium phosphate cement (CPC) was prepared. The setting time, pH value, compressive strength, X-ray diffraction (XRD) and scanning electron microscope (SEM) analysis of CPC were studied. The results show that TTCP containing small amount of CaO can be successfully attained heated at 1 500 degrees C for 6 h in vacuum condition. The initial setting time and final setting time of CPC is 4 min and 15 min, respectively. Its compressive strength is 20 MPa after 1-day immersion and 35 MPa after 7-day immersion. The pH value of the solution changes between 6.4 and 8.9. These properties can satisfy the clinical requirements of CPC. The final product of CPC is flake-like or needle-like hydroxyapatite (Ca5(P04)3OH, HA). The continuous network structure of HA appears in the microstructure, this leads to increase the strength of the material. This CPC can be used as bone substitute material.

Nonallelic interactions between het-c and a polymorphic locus, pin-c, are essential for nonself recognition and programmed cell death in Neurospora crassa.

Nonself recognition in filamentous fungi is conferred by genetic differences at het (heterokaryon incompatibility) loci. When individuals that differ in het specificity undergo hyphal fusion, the heterokaryon undergoes a programmed cell death reaction or is highly unstable. In Neurospora crassa, three allelic specificities at the het-c locus are conferred by a highly polymorphic domain. This domain shows trans-species polymorphisms indicative of balancing selection, consistent with the role of het loci in nonself recognition. We determined that a locus closely linked to het-c, called pin-c (partner for incompatibility with het-c) was required for het-c nonself recognition and heterokaryon incompatibility (HI). The pin-c alleles in isolates that differ in het-c specificity were extremely polymorphic. Heterokaryon and transformation tests showed that nonself recognition was mediated by synergistic nonallelic interactions between het-c and pin-c, while allelic interactions at het-c increased the severity of the HI phenotype. The pin-c locus encodes a protein containing a HET domain; predicted proteins containing HET domains are frequent in filamentous ascomycete genomes. These data suggest that nonallelic interactions may be important in nonself recognition in filamentous fungi and that proteins containing a HET domain may be a key factor in these interactions.

The control of mating type heterokaryon incompatibility by vib-1, a locus involved in het-c heterokaryon incompatibility in Neurospora crassa.

The mating-type (mat) locus of Neurospora crassa has a dual function. It is required for mating during sexual development, but it also mediates heterokaryon incompatibility following hyphal fusion during vegetative growth. Previously, it was determined that mutations in vib-1, which encodes a putative transcriptional regulator, suppress het-c heterokaryon incompatibility. This study showed that mutations in vib-1 suppress mat heterokaryon incompatibility in A/a partial diploids; suppression in (A+a) heterokaryons was variable and affected by unknown genetic factors. tol, a gene required for mat heterokaryon incompatibility, is epistatic to vib-1 in mediating mat heterokaryon incompatibility. vib-1 and tol also display a hierarchical order of strength of heterokaryon incompatibility that affected fertilization patterns. In addition to its role in heterokaryon incompatibility, vib-1 also negatively regulates conidiation. This study shows that FL, a positive regulator of conidiation, may partially activate conidiation by repressing VIB-1 function. VIB-1 may also partially regulate heterokaryon incompatibility mediated by genetic differences at het-e and het-8 because mutations in vib-1 increased the survival rate of self-incompatible het-8 and het-e partial diploid progeny.

Chromosome rearrangements in isolates that escape from het-c heterokaryon incompatibility in Neurospora crassa.

Chromosomal rearrangement is implicated in human cancers and hereditary diseases. Mechanisms generating chromosomal rearrangements may be shared by a variety of organisms. Spontaneous chromosomal rearrangements, especially large deletions, take place at high frequency in isolates that escape from heterokaryon incompatibility in Neurospora crassa. In this study, chromosomal rearrangements were detected in strains that had escaped from het-c heterokaryon incompatibility in N. crassa. A vc1 mutant carried a 20-kbp deletion covering five ORFs. A vc2 mutant carried a complex chromosome rearrangement with an 8-kbp deletion covering three ORFs, a 34-bp deletion and an 80-kbp inversion. The break-points of chromosome rearrangements in the vc1 and vc2 mutants all have direct repeats of 2 bp, similar to the break-points of some chromosome rearrangements associated with human cancer and genetic diseases. An ahc mutant carried a 31-kbp deletion covering at least 11 ORFs and a het-c deletion mutant carried a 7-kbp deletion covering two ORFs. Additional chromosomal rearrangements occurred in these two strains. These results indicate that escape from heterokaryon incompatibility can be used as a model system for chromosome rearrangement and DNA-repair studies. The impact of the chromosomal rearrangements is discussed, especially the deletion of the predicted ORFs on the phenotype of mutants.

Identification of vib-1, a locus involved in vegetative incompatibility mediated by het-c in Neurospora crassa.

A non-self-recognition system called vegetative incompatibility is ubiquitous in filamentous fungi and is genetically regulated by het loci. Different fungal individuals are unable to form viable heterokaryons if they differ in allelic specificity at a het locus. To identify components of vegetative incompatibility mediated by allelic differences at the het-c locus of Neurospora crassa, we isolated mutants that suppressed phenotypic aspects of het-c vegetative incompatibility. Three deletion mutants were identified; the deletions overlapped each other in an ORF named vib-1 (vegetative incompatibility blocked). Mutations in vib-1 fully relieved growth inhibition and repression of conidiation conferred by het-c vegetative incompatibility and significantly reduced hyphal compartmentation and death rates. The vib-1 mutants displayed a profuse conidiation pattern, suggesting that VIB-1 is a regulator of conidiation. VIB-1 shares a region of similarity to PHOG, a possible phosphate nonrepressible acid phosphatase in Aspergillus nidulans. Native gel analysis of wild-type strains and vib-1 mutants indicated that vib-1 is not the structural gene for nonrepressible acid phosphatase, but rather may regulate nonrepressible acid phosphatase activity.

The ham-2 locus, encoding a putative transmembrane protein, is required for hyphal fusion in Neurospora crassa.

Somatic cell fusion is common during organogenesis in multicellular eukaryotes, although the molecular mechanism of cell fusion is poorly understood. In filamentous fungi, somatic cell fusion occurs during vegetative growth. Filamentous fungi grow as multinucleate hyphal tubes that undergo frequent hyphal fusion (anastomosis) during colony expansion, resulting in the formation of a hyphal network. The molecular mechanism of the hyphal fusion process and the role of networked hyphae in the growth and development of these organisms are unexplored questions. We use the filamentous fungus Neurospora crassa as a model to study the molecular mechanism of hyphal fusion. In this study, we identified a deletion mutant that was restricted in its ability to undergo both self-hyphal fusion and fusion with a different individual to form a heterokaryon. This deletion mutant displayed pleiotropic defects, including shortened aerial hyphae, altered conidiation pattern, female sterility, slow growth rate, lack of hyphal fusion, and suppression of vegetative incompatibility. Complementation with a single open reading frame (ORF) within the deletion region in this mutant restored near wild-type growth rates, female fertility, aerial hyphae formation, and hyphal fusion, but not vegetative incompatibility and wild-type conidiation pattern. This ORF, which we named ham-2 (for hyphal anastomosis), encodes a putative transmembrane protein that is highly conserved, but of unknown function among eukaryotes.