Phytophthora : taxonomic and phylogenetic revision of the genus.

Many members of the Oomycota genus Phytophthora cause economic and environmental impact diseases in nurseries, horticulture, forest, and natural ecosystems and many are of regulatory concern around the world. At present, there are 223 described species, including eight unculturable and three lost species. Twenty-eight species need to be redescribed or validated. A lectotype, epitype or neotype was selected for 20 species, and a redescription based on the morphological/molecular characters and phylogenetic placement is provided. In addition, the names of five species are validated: P. cajani, P. honggalleglyana (Synonym: P. hydropathica), P. megakarya, P. pisi and P. pseudopolonica for which morphology and phylogeny are given. Two species, P. ×multiformis and P. uniformis are presented as new combinations. Phytophthora palmivora is treated with a representative strain as both lecto- and epitypification are pending. This manuscript provides the updated multigene phylogeny and molecular toolbox with seven genes (ITS rDNA, ß-tub, COI, EF1α, HSP90, L10, and YPT1) generated from the type specimens of 212 validly published, and culturable species (including nine hybrid taxa). The genome information of 23 types published to date is also included. Several aspects of the taxonomic revision and phylogenetic re-evaluation of the genus including species concepts, concept and position of the phylogenetic clades recognized within Phytophthora are discussed. Some of the contents of this manuscript, including factsheets for the 212 species, are associated with the "IDphy: molecular and morphological identification of Phytophthora based on the types" online resource (https://idtools.org/tools/1056/index.cfm). The first version of the IDphy online resource released to the public in September 2019 contained 161 species. In conjunction with this publication, we are updating the IDphy online resource to version 2 to include the 51 species recently described. The current status of the 223 described species is provided along with information on type specimens with details of the host (substrate), location, year of collection and publications. Additional information is provided regarding the ex-type culture(s) for the 212 valid culturable species and the diagnostic molecular toolbox with seven genes that includes the two metabarcoding genes (ITS and COI) that are important for Sanger sequencing and also very valuable Molecular Operational Taxonomic Units (MOTU) for second and third generation metabarcoding High-throughput sequencing (HTS) technologies. The IDphy online resource will continue to be updated annually to include new descriptions. This manuscript in conjunction with IDphy represents a monographic study and the most updated revision of the taxonomy and phylogeny of Phytophthora, widely considered one of the most important genera of plant pathogens. Taxonomic novelties: New species: Phytophthora cajani K.S. Amin, Baldev & F.J. Williams ex Abad, Phytophthora honggalleglyana Abad, Phytophthora megakarya Brasier & M.J. Griffin ex Abad, Phytophthora pisi Heyman ex Abad, Phytophthora pseudopolonica W.W. Li, W.X. Huai & W.X. Zhao ex Abad & Kasiborski; New combinations: Phytophthora ×multiformis (Brasier & S.A. Kirk) Abad, Phytophthora uniformis (Brasier & S.A. Kirk) Abad; Epitypifications (basionyms): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne, Phytophthora ilicis Buddenh. & Roy A. Young, Phytophthora inundata Brasier et al., Phytophthora megasperma Drechsler, Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Lectotypifications (basionym): Peronospora cactorum Lebert & Cohn, Pythiacystis citrophthora R.E. Sm. & E.H. Sm., Phytophthora colocasiae Racib., Phytophthora drechsleri Tucker, Phytophthora erythroseptica Pethybr., Phytophthora fragariae Hickman, Phytophthora hibernalis Carne, Phytophthora ilicis Buddenh. & Roy A. Young, Phytophthora megasperma Drechsler, Phytophthora mexicana Hotson & Hartge, Phytophthora nicotianae Breda de Haan, Phytophthora phaseoli Thaxt., Phytophthora porri Foister, Phytophthora primulae J.A. Toml., Phytophthora sojae Kaufm. & Gerd., Phytophthora vignae Purss, Pythiomorpha gonapodyides H.E. Petersen; Neotypifications (basionym): Phloeophthora syringae Kleb., Phytophthora meadii McRae Citation: Abad ZG, Burgess TI, Bourret T, Bensch K, Cacciola S, Scanu B, Mathew R, Kasiborski B, Srivastava S, Kageyama K, Bienapfl JC, Verkleij G, Broders K, Schena L, Redford AJ (2023). Phytophthora: taxonomic and phylogenetic revision of the genus. Studies in Mycology 106: 259-348. doi: 10.3114/sim.2023.106.05.

Destabilization of tumor necrosis factor-alpha mRNA by 5-alpha dihydrotestosterone in Jurkat cells.

The effect of a male steroid hormone, 5DHT, on the expression of TNF-alpha was examined using a human leukemia T cell line, Jurkat. Cells were treated with 5DHT in the presence or absence of PHA, and RNA was isolated followed by a reverse transcriptase - mediated PCR (RT-PCR) to measure the steady state levels of TNF-alpha mRNA. The treatment of cells with 5DHT resulted in a 50% of decrease in the level of TNF-alpha mRNA compared to that in untreated conditions (basal level). A similar level of reduction of the message by 5DHT was also observed in PHA-stimulated cells. The reduction of the steady state levels of TNF-alpha mRNA in Jurkat cells was a result of destabilization of the gene as demonstrated by actinomycin D treatment; a half-life of TNF-alpha message in 5DHT treated cells and non-treated cells was 1 hr and 2.5 hr, respectively, whereas that in 5DHT/PHA and PHA-treated cells was 3hr and 6hr, respectively.

Methylation status of 5'-regulatory region of tumor necrosis factor alpha gene correlates with differentiation stages of monocytes.

The DNA methylation status of the 5'-regulatory region of human tumor necrosis factor-alpha (TNF-alpha) gene was examined using two human monocytic cell lines representing different differentiation stages: one cell line, THP-1, represents the most differentiated macrophage-like phenotypes and the other, HL-60, represents a least differentiated phenotype. Two restriction enzymes were used to detect methylated sites in the 5'-regulatory region of the TNF-alpha gene. The restriction enzyme Msp I, which recognizes both CCGG (unmethylated) and C(me)CGG (methylated) sequences, whereas Hpa II recognizes only the unmethylated CCGG sequence. Two Msp I sites located at -600 and +200 nucleotides, respectively, from the transcriptional initiation site were unmethylated in DNA from the THP-1, whereas these sites were methylated in the one from the HL-60. Treating the HL-60 cells with 1,25-vitamin D3, ( 1,25-(OH)2D3), which is known to induce monocytic cell differentiation, induce demethylation of the sites, suggesting the methylation status was correlated with the differentiation stages of monocytes.

In situ islet cytokine gene expression during development of type I diabetes in the non-obese diabetic mouse.

Expression of cytokine genes in the islets of non-obese diabetic (NOD) female mice was examined. RNA samples were prepared from the islets and spleens of NOD mice at different time points at prediabetic stages during the natural disease process. Cytofluorometric analyses showed that the majority of lymphocyte infiltrates in the islets at 14 weeks of age consisted of T cells (68%). Of these, 80% of Thy1.2+ cells were CD4+ T cells. Less than 1% of in situ islet immune cells expressed a cell surface marker specific for the macrophage (Mac-1). Results of polymerase chain reaction using RNA (RT-PCR) prepared from spleens, and isolated and purified islets demonstrated that IFN-gamma message was detectable in the islets at 7 weeks of age (an early stage of insulitis). No message for this gene was detected in the spleen at any stage studied (7, 14 and 16 weeks of age). In contrast, TNF-alpha message was detected in both spleen and the islets at all stages, although the level of expression of TNF-alpha in the islets was much higher than that in the spleen. These results suggest that both cytokines are produced by in situ islet T cells, possibly activated T cells, which may be responsible for initiating or perpetuating autoimmune reactions in the islets.

Distinct associations of HLA class II genes with inflammatory bowel disease.

BACKGROUND: There are relatively few studies of HLA class II association either with Crohn's disease (CD) or ulcerative colitis (UC). The few available association studies have been carried out by serological techniques, and the results from these studies are inconclusive. METHODS: The association between HLA class II genes was studied using molecular genotyping in combination with allele-specific oligonucleotide hybridization by polymerase chain reactions. RESULTS: In UC (n = 74), we observed a positive association with the HLA DR2 allele (P = 0.008) and negative associations with the DR4 (P = 0.018) and DRw6 (P = 0.028) when compared with ethnically matched controls (n = 77). No associations were observed with any DQ alleles. In contrast, in CD (n = 95) we observed a positive association with the combination of DR1 and DQw5 alleles (P = 0.021). Furthermore, stratifying DR1 and DQw5 alleles indicated that neither allele was independently associated with CD, suggesting that the association was with the haplotype rather than either of the alleles individually. A suballele of DQw5, DQB1*0501, contributed this haplotypic association (P = 0.012). CONCLUSIONS: DR and DQ molecules firmly separate UC and CD on genetic grounds, suggesting that the contribution of the HLA class II genes to the disease susceptibility is quite different for the two disorders.

In situ islet T cell receptor variable region gene usage in the nonobese diabetic mouse.

Several features of the genetics and immunopathology of diabetes in the nonobese diabetic (NOD) mouse, which spontaneously develops type I diabetes, are shared with the human disease. Immunohistochemical studies support the concept that T lymphocytes are the major components of inflammatory cells in the pancreatic islets and these cells may play a critical role in the destruction of the beta cells leading to diabetes. Therefore, we examined whether particular TCR-beta variable region genes were utilized by in situ islet T cells at different stages (4 - 5, 7, 14 - 15 and 16 weeks of age) of the disease process. Dot-blot hybridization was performed using RNA prepared from isolated islets, thymus, spleen, peripheral blood leukocytes and axillary lymph nodes of 10 to 15 mice pooled for each data point. Ten different TCR V-beta probes were used for the analyses. Limited usage of islet V-beta genes was observed only at the early prediabetic stage (4 - 5 weeks old) of the disease. At later stages of the disease (7 - 16 weeks old), no preferential usage of TCR genes was observed in the islets compared to those of peripheral lymphoid organs. These data suggest that only certain types of T cells bearing particular TCR V-beta genes may be responsible for initiating and perpetuating infiltration of immune cells into the islets and these particular T cells are only identified at the very early stages of the autoimmune process.

Allele-specific methylation in the 5'-regulatory region of class II DQ beta genes in the human major histocompatibility complex (MHC): relationship to autoimmune disease susceptibility.

The DNA methylation state of the 5'-regulatory region of human HLA-DQ beta genes was examined. Two restriction enzymes were utilized to detect methylated (meCG) dinucleotides in the 5'-regulatory region of the DQ-beta genes: the restriction enzyme Msp I, which recognizes CCGG and CmeCGG, and Hpa II recognizes only the unmethylated CG sequence. DNA samples were prepared from 95 HLA-typed individuals including 40 B-lymphoblastoid cell lines and peripheral blood leukocytes of 55 individuals. Of these samples, 20 were from parents of individuals with insulin-dependent diabetes. Allele specific methylation was observed in particular DR-associated DQ-beta gene alleles. The DQw8 (DQw3.2) allele, most DQw7 (DQw3.1) alleles, and the DR3-associated DQw2 allele were all unmethylated. The parental methylation state was stably transmitted to offspring. Because these DQ alleles are highly associated with several autoimmune diseases, our results raise the possibility that the regulation of expression of these particular DQ-beta alleles might be different from that of other alleles, and that the 5'-regulatory DNA sequences of these particular DQ beta alleles may be responsible for, or contribute to, susceptibility to autoimmune diseases.

Restriction fragment length polymorphism (RFLP) heterogeneity of HLA-DQ beta genes associated with DNA fragment identical to the DR1-beta DNA structure.

Restriction fragment length polymorphism (RFLP) analyses of DR1 positive peripheral blood leukocytes DNA was carried out. The Taq I digested DNA was hybridized with cDNA probes for HLA-DR and -DQ beta genes. The DR probe detected fragments commonly observed in the DR1 specificity, whereas a new DQ-beta fragment was detected in some DR1 haplotypes when the DQ-beta probe was used. This fragment had an RFLP pattern identical to the DQ-beta fragment typically associated with most DR2 and some DRw6 specificities.