Development of secondary mutations in wild-type and mutant EZH2 alleles cooperates to confer resistance to EZH2 inhibitors.

The histone methyltransferase Enhancer of Zeste Homolog 2 (EZH2) is frequently dysregulated in cancers, and gain-of-function (GOF) EZH2 mutations have been identified in non-Hodgkin lymphomas. Small-molecule inhibitors against EZH2 demonstrated anti-tumor activity in EZH2-mutated lymphomas and entered clinical trials. Here, we developed models of acquired resistance to EZH2 inhibitor EI1 with EZH2-mutated lymphoma cells. Resistance was generated by secondary mutations in both wild-type (WT) and GOF Y641N EZH2 alleles. These EZH2 mutants retained the substrate specificity of their predecessor complexes but became refractory to biochemical inhibition by EZH2 inhibitors. Resistant cells were able to maintain a high level of H3K27Me3 in the presence of inhibitors. Interestingly, mutation of EZH2 WT alone generated an intermediate resistance phenotype, which is consistent with a previously proposed model of cooperation between EZH2 WT and Y641N mutants to promote tumorigenesis. In addition, the findings presented here have implications for the clinical translation of EZH2 inhibitors and underscore the need to develop novel EZH2 inhibitors to target potential resistance emerging in clinical settings.

Colletotrichum gloeosporioides pelB is an important virulence factor in avocado fruit-fungus interaction.

Colletotrichum gloeosporioides is an important pathogen of tropical and subtropical fruits. The C. gloeosporioides pelB gene was disrupted in the fungus via homologous recombination. Three independent isolates, GD-14, GD-23, and GD-29, did not produce or secrete pectate lyase B (PLB) and exhibited 25% lower pectate lyase (PL) and pectin lyase (PNL) activities and 15% higher polygalacturonase (PG) activity than the wild type. The PLB mutants exhibited no growth reduction on glucose, Na polypectate, or pectin as the sole carbon source at pH 3.8 or 6.0, except for a 15% reduction on pectin at pH 6.0. When pelB mutants were inoculated onto avocado fruits, however, a 36 to 45% reduction in estimated decay diameter was observed compared with the two controls, the wild type and undisrupted transformed isolate. In addition, these pelB mutants induced a significantly higher host phenylalanine ammonia lyase activity as well as the antifungal diene, which is indicative of higher host resistance. These results suggest that PLB is an important factor in the attack of C. gloeosporioides on avocado fruit, probably as a result of its virulence factor and role in the induction of host defense mechanisms.

Microbial phyllosphere populations are more complex than previously realized.

Phyllosphere microbial communities were evaluated on leaves of field-grown plant species by culture-dependent and -independent methods. Denaturing gradient gel electrophoresis (DGGE) with 16S rDNA primers generally indicated that microbial community structures were similar on different individuals of the same plant species, but unique on different plant species. Phyllosphere bacteria were identified from Citrus sinesis (cv. Valencia) by using DGGE analysis followed by cloning and sequencing of the dominant rDNA bands. Of the 17 unique sequences obtained, database queries showed only four strains that had been described previously as phyllosphere bacteria. Five of the 17 sequences had 16S similarities lower than 90% to database entries, suggesting that they represent previously undescribed species. In addition, three fungal species were also identified. Very different 16S rDNA DGGE banding profiles were obtained when replicate cv. Valencia leaf samples were cultured in BIOLOG EcoPlates for 4.5 days. All of these rDNA sequences had 97--100% similarity to those of known phyllosphere bacteria, but only two of them matched those identified by the culture independent DGGE analysis. Like other studied ecosystems, microbial phyllosphere communities therefore are more complex than previously thought, based on conventional culture-based methods.

Preventive service utilisation as a predictor for emergency dental examinations.

OBJECTIVE: To quantify the relationship between receipt of routine dental care and the use of non-trauma related emergency dental services. DESIGN: A multiple logistic regression was run on administrative dental claim and encounter data. The model dependent variable was the use of non-trauma related emergency dental care. Predictors included previous year oral examinations, radiographs, dental cleanings and, as a control, member age. SETTING: Administrative data were obtained from a dental health maintenance organisation located in the state of Texas. SUBJECTS: Claim and encounter data for 2,947 insured members were used, representing experience from 1995 through 1996. OUTCOME MEASURES: The outcome of interest was the use of non-trauma related emergency dental services. RESULTS: Results demonstrated empirically that those who availed themselves of preventive dental services were significantly less likely to use non-trauma related emergency services (P<0.01). The probability of needing non-trauma related dental services in 1996 was 42.7% lower among those who had an examination in 1995 when compared with those who did not. When analysed in a simple logistic regression, dental cleanings in 1995 were also significantly associated with a decreased probability of needing non-trauma related emergency services. However, this relationship did not hold in the controlled model, which was probably due to multicollinearity. CONCLUSIONS: This study provides evidence of the value of periodic preventive dental examinations and services. Those who receive such services are less likely to use non-trauma related emergency dental services.

Expression of pectate lyase from Colletotrichum gloesosporioides in C. magna promotes pathogenicity.

To test the contribution of pectate lyase (PL) to promoting fungal pathogenicity, a pectate lyase gene (pel) from the avocado pathogen Colletotrichum gloeosporioides, isolate Cg-14, was expressed in C. magna isolate L-2.5, a pathogen of cucurbits that causes minor symptoms in watermelon seedlings and avocado fruits. Isolate L-2.5 was transformed with pPCPH-1 containing hph-B as a selectable marker and the 4.1-kb genomic pel clone. Southern hybridization, with the 4.1-kb genomic pel clone or 2.13-kb hph-B cassette as probes, detected integration of pel in transformed C. magna isolates Cm-PL-3 and Cm-PL-10. Western blot (immunoblot) analysis with antibodies against Cg-14 PL detected a single PL secreted by L-2.5 at a molecular mass of 41.5 kDa, whereas the PL of C. gloeosporioides had a molecular mass of 39 kDa. When PL activity was measured 4 days after inoculation in pectolytic enzyme-inducing media (PEIM), transformed isolates Cm-PL-3 and Cm-PL-10 showed additive PL activity relative to both Cg-14 and L-2.5. Transformed isolates also showed additive maceration capabilities on avocado pericarp relative to the wild-type C. magna alone, but did not reach the maceration ability of C. gloeosporioides. However, more severe maceration and damping off developed in watermelon seedlings inoculated with the transformed isolates compared with the two wild-type isolates, which showed no symptom development on these seedlings during the same period. Results clearly show the contribution of a single pel to the pathogenic abilities of C. magna and suggest that PL is a pathogenicity factor required for the penetration and colonization of Colletotrichum species.

From rags to riches: insights from the first genomic sequence of a plant pathogenic bacterium.

The recently published genomic sequence of Xylella fastidiosa is the first for a free-living plant pathogen and provides clues to mechanisms of pathogenesis and survival in insect vectors. The sequence data should lead to improved control of this pathogen.

A Century of Plant Pathology: A Retrospective View on Understanding Host-Parasite Interactions.

▪ Abstract The twentieth century has been productive for the science of plant pathology and the field of host-parasite interactions-both in understanding how pathogens and plant defense work and in developing more effective means of disease control. Early in the twentieth century, plant pathology adopted a philosophy that encouraged basic scientific investigation of pathogens and disease defense. That philosophy led to the strategy of developing disease-resistant plants as a prima facie disease-control measure-and in the process saved billions of dollars and avoided the use of tons of pesticides. Plant pathology rapidly adopted molecular cloning and its spin-off technologies, and these have fueled major advances in our basic understanding of plant diseases. This knowledge and the development of efficient technologies for producing transgenic plants convey optimism that plant diseases will be more efficiently controlled in the twenty-first century.

Structure of a plant cell wall fragment complexed to pectate lyase C.

The three-dimensional structure of a complex between the pectate lyase C (PelC) R218K mutant and a plant cell wall fragment has been determined by x-ray diffraction techniques to a resolution of 2.2 A and refined to a crystallographic R factor of 18.6%. The oligosaccharide substrate, alpha-D-GalpA-([1-->4]-alpha-D-GalpA)3-(1-->4)-D-GalpA , is composed of five galacturonopyranose units (D-GalpA) linked by alpha-(1-->4) glycosidic bonds. PelC is secreted by the plant pathogen Erwinia chrysanthemi and degrades the pectate component of plant cell walls in soft rot diseases. The substrate has been trapped in crystals by using the inactive R218K mutant. Four of the five saccharide units of the substrate are well ordered and represent an atomic view of the pectate component in plant cell walls. The conformation of the pectate fragment is a mix of 21 and 31 right-handed helices. The substrate binds in a cleft, interacting primarily with positively charged groups: either lysine or arginine amino acids on PelC or the four Ca2+ ions found in the complex. The observed protein-oligosaccharide interactions provide a functional explanation for many of the invariant and conserved amino acids in the pectate lyase family of proteins. Because the R218K PelC-galacturonopentaose complex represents an intermediate in the reaction pathway, the structure also reveals important details regarding the enzymatic mechanism. Notably, the results suggest that an arginine, which is invariant in the pectate lyase superfamily, is the amino acid that initiates proton abstraction during the beta elimination cleavage of polygalacturonic acid.

Characterization of a 34-kDa soybean binding protein for the syringolide elicitors.

Syringolides are water-soluble, low-molecular-weight elicitors that trigger defense responses in soybean cultivars carrying the Rpg4 disease-resistance gene but not in rpg4 cultivars. 125I-syringolide 1 previously was shown to bind to a soluble protein(s) in extracts from soybean leaves. A 34-kDa protein that accounted for 125I-syringolide 1 binding activity was isolated with a syringolide affinity-gel column. Partial sequences of internal peptides of the 34-kDa protein were identical to P34, a previously described soybean seed allergen. In soybean seeds, P34 is processed from a 46-kDa precursor protein and was shown to have homology with thiol proteases. P34 is a moderately abundant protein in soybean seeds and cotyledons but its level in leaves is low. cDNAs encoding 46-, 34-, and 32-kDa forms of the soybean protein were cloned into the baculovirus vector, pVL1392, and expressed in insect cells. The resulting 32- and 34-kDa proteins, but not the 46-kDa protein, exhibited ligand-specific 125I-syringolide binding activity. These results suggest that P34 may be the receptor that mediates syringolide signaling.

External loops at the C terminus of Erwinia chrysanthemi pectate lyase C are required for species-specific secretion through the out type II pathway.

The type II secretion system (main terminal branch of the general secretion pathway) is used by diverse gram-negative bacteria to secrete extracellular proteins. Proteins secreted by this pathway are synthesized with an N-terminal signal peptide which is removed upon translocation across the inner membrane, but the signals which target the mature proteins for secretion across the outer membrane are unknown. The plant pathogens Erwinia chrysanthemi and Erwinia carotovora secrete several isozymes of pectate lyase (Pel) by the out-encoded type II pathway. However, these two bacteria cannot secrete Pels encoded by heterologously expressed pel genes from the other species, suggesting the existence of species-specific secretion signals within these proteins. The functional cluster of E. chrysanthemi out genes carried on cosmid pCPP2006 enables Escherichia coli to secrete E. chrysanthemi, but not E. carotovora, Pels. We exploited the high sequence similarity between E. chrysanthemi PelC and E. carotovora Pel1 to construct 15 hybrid proteins in which different regions of PelC were replaced with homologous sequences from Pell. The differential secretion of these hybrid proteins by E. coli(pCPP2006) revealed M118 to D175 and V215 to C329 as regions required for species-specific secretion of PelC. We propose that the primary targeting signal is contained within the external loops formed by G274 to C329 but is dependent on residues in M118 to D170 and V215 to G274 for proper positioning.

Accumulation of salicylic acid and 4-hydroxybenzoic acid in phloem fluids of cucumber during systemic acquired resistance is preceded by a transient increase in phenylalanine ammonia-lyase activity in petioles and stems.

Cucumber (Cucumis sativa) leaves infiltrated with Pseudomonas syringae pv. syringae cells produced a mobile signal for systemic acquired resistance between 3 and 6 h after inoculation. The production of a mobile signal by inoculated leaves was followed by a transient increase in phenylalanine ammonia-lyase (PAL) activity in the petioles of inoculated leaves and in stems above inoculated leaves; with peaks in activity at 9 and 12 h, respectively, after inoculation. In contrast, PAL activity in inoculated leaves continued to rise slowly for at least 18 h. No increases in PAL activity were detected in healthy leaves of inoculated plants. Two benzoic acid derivatives, salicylic acid (SA) and 4-hydroxybenzoic acid (4HBA), began to accumulate in phloem fluids at about the time PAL activity began to increase, reaching maximum concentrations 15 h after inoculation. The accumulation of SA and 4HBA in phloem fluids was unaffected by the removal of all leaves 6 h after inoculation, and seedlings excised from roots prior to inoculation still accumulated high levels of SA and 4HBA. These results suggest that SA and 4HBA are synthesized de novo in stems and petioles in response to a mobile signal from the inoculated leaf.

Transfer of Tat and release of TAR RNA during the activation of the human immunodeficiency virus type-1 transcription elongation complex.

The HIV-1 trans-activator protein, Tat, is a potent activator of transcriptional elongation. Tat is recruited to the elongating RNA polymerase during its transit through the trans-activation response region (TAR) because of its ability to bind directly to TAR RNA expressed on the nascent RNA chain. We have shown that transcription complexes that have acquired Tat produce 3-fold more full-length transcripts than complexes not exposed to Tat. Western blotting experiments demonstrated that Tat is tightly associated with the paused polymerases. To determine whether TAR RNA also becomes attached to the transcription complex, DNA oligonucleotides were annealed to the nascent chains on the arrested complexes and the RNA was cleaved by RNase H. After cleavage, the 5' end of the nascent chain, carrying TAR RNA, is quantitatively removed, but the 3' end of the transcript remains associated with the transcription complex. Even after the removal of TAR RNA, transcription complexes that have been activated by Tat show enhanced processivity. We conclude that Tat, together with cellular co-factors, becomes attached to the transcription complex and stimulates processivity, whereas TAR RNA does not play a direct role in the activation of elongation and is used simply to recruit Tat and cellular co-factors.

Specific Binding of the Syringolide Elicitors to a Soluble Protein Fraction from Soybean Leaves.

Syringolides are glycolipid elicitors produced by Gram-negative bacteria expressing Pseudomonas syringae avirulence gene D. The syringolides mediate gene-for-gene complementarity, inducing the hypersensitive response only in soybean plants carrying the Rpg4 disease resistance gene. A site(s) for 125I-syringolide 1 was detected in the soluble protein fraction from soybean leaves, but no evidence for ligand-specific binding to the microsomal fraction was obtained. The Kd value for syringolide 1 binding with the soluble fraction was 8.7 nM, and binding was greatly reduced by prior protease treatment or heating. A native gel assay was also used to demonstrate ligand-specific binding of labeled syringolide 1 with a soluble protein(s). Competition studies with 125I-syringolide 1 and several structural derivatives demonstrated a direct correlation between binding affinity to the soluble fraction and elicitor activity. However, differential competition binding studies disclosed no differences in syringolide binding to soluble fractions from Rpg4/Rpg4 or rpg4/rpg4 soybean leaves. Thus, the observed binding site fulfills several criteria expected of an intracellular receptor for the syringolides, but it is most likely not encoded by the Rpg4 gene. Instead, the Rpg4 gene product may function subsequent to elicitor binding, possibly in intracellular signal transduction.

Comparison of avrD alleles from Pseudomonas syringae pv. glycinea.

Avirulence gene D alleles resided on indigenous plasmids in races 0, 2, 3, 4, 5, and 6 of Pseudomonas syringae pv. glycinea (Psg), but the allele in race 1 appeared to be chromosomal. These were all nonfunctional avirulence genes because they neither induced the avirulence phenotype on Rpg4 soybean cultivars nor directed the production of syringolide elicitors when expressed in Escherichia coli cells. The predicted proteins encoded by the seven Psg avrD genes were very similar to that of a functional class II allele from P. syringae pv. phaseolicola G50 race 2, but contained mutations collectively affecting only nine amino acid positions. Despite these relatively small amino acid differences and the location of avrD from each isolate on a 5.6-kb HindIII restriction fragment, the flanking regions varied considerably among the Psg isolates. The presence of avrD alleles with few alterations but different locational contexts in all tested Psg races argues that they provide an important selected function in the bacteria but have been modified to escape defense surveillance in Rpg4 soybean plants.

Cloning, sequence and expression of the pel gene from an Amycolata sp.

The pel gene from an Amycolata sp. encoding a pectate lyase (EC 4.2.2.2) was isolated by activity screening a genomic DNA library in Streptomyces lividans TK24. Subsequent subcloning and sequencing of a 2.3 kb BamHI BglII fragment revealed an open reading frame of 930 nt corresponding to a protein of 29,660 Da. The overall G + C content for the coding region was 65%, with a strong G + C preference in the third (wobble) codon position (93%). A putative ribosome-binding site 5'-GGGAG-3' preceded the translational start codon by 7 base pairs. The Amycolata pectate lyase contains a signal peptide of 26 amino acids, that is cleaved after the sequence Ala-Thr-Ala. The size of the deduced protein as well as its N-terminal amino-acid sequence match the wild-type pectate lyase from the Amycolata sp. Expression of the pel gene in S. lividans TK24 resulted in high pectate lyase activity in the culture supernatant, concomitant with the appearance of a dominant protein band on a sodium dodecyl polyacrylamide gel at 30 kDa. No pectate lyase activity was detected in E. coli BL21 with the pel gene under the strong T7 promotor. The deduced amino-acid sequence showed 40% identity with PelE from Erwinia chrysanthemi and the pectate lyase from Glomerella cingulata. The Amycolata pectate lyase clearly belongs to the pectate lyase superfamily, sharing all functional amino acids and likely has a similar structural topology as Pels from Erwinia chrysanthemi and Bacillus subtilis.