Genome-wide analysis of R2R3-MYB transcription factors in poplar and functional validation of PagMYB147 in defense against Melampsora magnusiana.

MAIN CONCLUSION: Transcription of PagMYB147 was induced in poplar infected by Melampsora magnusiana, and a decline in its expression levels increases the host's susceptibility, whereas its overexpression promotes resistance to rust disease. Poplars are valuable tree species with diverse industrial and silvicultural applications. The R2R3-MYB subfamily of transcription factors plays a crucial role in response to biotic stresses. However, the functional studies on poplar R2R3-MYB genes in resistance to leaf rust disease are still insufficient. We identified 191 putative R2R3-MYB genes in the Populus trichocarpa genome. A phylogenetic analysis grouped poplar R2R3-MYBs and Arabidopsis R2R3-MYBs into 33 subgroups. We detected 12 tandem duplication events and 148 segmental duplication events, with the latter likely being the main contributor to the expansion of poplar R2R3-MYB genes. The promoter regions of these genes contained numerous cis-acting regulatory elements associated with response to stress and phytohormones. Analyses of RNA-Seq data identified a multiple R2R3-MYB genes response to Melampsora magnusiana (Mmag). Among them, PagMYB147 was significantly up-regulated under Mmag inoculation, salicylic acid (SA) and methyl jasmonate (MeJA) treatment, and its encoded product was primarily localized to the cell nucleus. Silencing of PagMYB147 exacerbated the severity of Mmag infection, likely because of decreased reactive oxygen species (ROS) production and phenylalanine ammonia-lyase (PAL) enzyme activity, and up-regulation of genes related to ROS scavenging and down-regulation of genes related to PAL, SA and JA signaling pathway. In contrast, plants overexpressing PagMYB147 showed the opposite ROS accumulation, PAL enzyme activity, SA and JA-related gene expressions, and improved Mmag resistance. Our findings suggest that PagMYB147 acts as a positive regulatory factor, affecting resistance in poplar to Mmag by its involvement in the regulation of ROS homeostasis, SA and JA signaling pathway.

Ontogenetic Variation in Macrocyclic and Hemicyclic Poplar Rust Fungi.

Melampsora larici-populina (Mlp), M. medusae (Mmed), M. magnusiana (Mmag), and M. pruinosae (Mpr) are epidemic rust fungi in China. The first two are macrocyclic rust fungi distributed in temperate humid environments. The latter two are hemicyclic rusts, mainly distributed in arid and semi-arid areas. Ontogenetic variation that comes with this arid-resistance is of great interest-and may help us predict the influence of a warmer, drier, climate on fungal phylogeny. To compare the differences in the life history and ontogeny between the two types of rust, we cloned mating type genes, STE3.4 and STE3.3 using RACE-smart technology. Protein structures, functions, and mutant loci were compared across each species. We also used microscopy to compare visible cytological differences at each life stage for the fungal species, looking for variation in structure and developmental timing. Quantitative PCR technology was used to check the expression of nuclear fusion and division genes downstream of STE3.3 and STE3.4. Encoding amino acids of STE3.3 and STE3.4 in hemicyclic rusts are shorter than these in the macrocyclic rusts. Both STE3.3 and STE3.4 interact with a protein kinase superfamily member EGG12818 and an E3 ubiquitin protein ligase EGG09709 directly, and activating G-beta conformational changes. The mutation at site 74th amino acid in the conserved transmembrane domain of STE3.3 ascribes to a positive selection, in which alanine (Ala) is changed to phenylalanine (Phe) in hemicyclic rusts, and a mutation with Tyr lost at site 387th in STE3.4, where it is the binding site for ß-D-Glucan. These mutants are speculated corresponding to the insensitivity of hemicyclic rust pheromone receptors to interact with MFa pheromones, and lead to Mnd1 unexpressed in teliospora, and they result in the diploid nuclei division failure and the sexual stage missing in the life cycle. A Phylogenic tree based on STE3.4 gene suggests these two rust types diverged about 14.36 million years ago. Although these rusts share a similar uredia and telia stage, they show markedly different wintering strategies. Hemicyclic rusts overwinter in the poplar buds endophytically, their urediniospores developing thicker cell walls. They form haustoria with a collar-like extrahaustorial membrane neck and induce host thickened callose cell walls, all ontogenetic adaptations to arid environments.

First Report of Leaf Rust of Acer mono Caused by Pucciniastrum hikosanense in China.

Acer mono Maxim, mainly distributed in China, Japan, Korea and eastern Russia (Shang et al. 2012), is a widely planted ornamental and pharmaceutical tree (Zhang et al. 2015). In September 2020, leaf samples of A. mono infected by uredinia were collected in Shaanxi Province (34°15'40.06'' N, 108°3'54.54'' E, alt. 432.35m), China. Telia development was observed in late autumn. Voucher specimens were deposited in the Herbarium Mycologicum Academiae Sinicae (no. HMAS249354), China. This led to premature defoliation and in the 90% planting wide incidence. Geospatial investigations revealed that this rust was widely distributed in local urban parks, but was nonpathogenic to A. buergerianum, A. negundo, A. oblongum, A. palmatum and A. rubrum. This fungus was morphologically characterized and most closely matched descriptions of Pucciniastrum. Uredinia were hypophyllous, subepidermal, scattered to gregarious, oval or round, 0.10-0.30 × 0.08-0.15 mm, golden yellow to orange, somewhat pulverulent. Peridia were hemispherical, erumpent with apical pores; peridial cells minute, irregularly polygonal, hyaline to pale yellow; ostiolar cells ellipsoid or roundish. Urediniospores were subglobose, ovate or ellipsoid, 20-33 × 15-21 µm, yellow to pale orange; wall 1-2 µm thick, hyaline to pale yellow, echinulate, somewhere smooth. Pedicels were deciduous, hyaline, minute, fragile. Telia were hypophyllous, subepidermal, intermixed with uredinia, irregularly polygonal, restricted by veins, 0.34-0.91 × 0.21-0.54 mm, and orange to amber brown. Teliospores were produced parallelly single-layered, and were subglobose, oblong, sometimes angular, 23-47 × 16-34 µm, colorless to pale yellow, 1-5 mediastinal, 2-6-celled; lateral wall 1-1.6 µm thick, apical wall 1-3 µm thick, smooth, hyaline. The internal transcribed spacer (ITS) and rDNA-28S regions were amplified using ITS1F/ITS4 and NL1/NL4 (Ji et al. 2019) to confirm the identification. The aligned sequences were deposited in GenBank (accession no. MW391829, MW543709, MW541916, MW541917). Phylogenetic trees were constructed based on neighbor-joining (NJ), maximum-likelihood (ML) and Bayesian methods. ML and NJ bootstrap values were calculated by bootstrap analyses of 1,000 replicates with GTR+G+I model using MEGA-X (Kumar et al. 2018), while Bayesian Markov chain Monte Carlo analyses were performed using MrBayes ver. 3.1.2 (Huelsenbeck & Ronquist 2001; Ji et al. 2019). Phylogenetic analysis revealed that HMAS249354 and Pucciniastrum hikosanense were grouped into one clade highly supported by bootstrap values of NJ, ML, and Bayesian posterior probability (Bpp) of 97%/93%/1, respectively. Koch's postulates were fulfilled with 1-year-old healthy plants of A. mono. Fresh urediniospores were collected and suspended in a 0.05% water solution of Tween 20, and 100 µl of urediniospores suspension (106 urediniospores/ml) per leaf (n=10) were sprayed, with another ten healthy leaves sprayed with sterile water as the control. The plants were placed in dark for 48 h and then moved into greenhouse at 22°C with 12 h light per day. Disease symptoms after 10-12 days' inoculation on the inoculated leaves which were identical to the original observations, while the control leaves remained healthy. Previously, P. hikosanense was reported to infect Acer rufinerve Sieb. et Zucc. in Japan (Hiratsuka 1940) and A. rubescens Hayata in Taiwan, China (Dai 1979). This is the first report of leaf rust of Acer mono caused by Pucciniastrum hikosanense Hirats. f. in China.

First report of alternate hosts of willow rust disease caused by Melampsora ferrinii in China.

Corydalis acuminata Franch., C. edulis Maxim. and C. racemosa (Thunb.) Pers. of family Papaveraceae are rich in multiple alkaloids and widely used as Chinese medicinal herbs, for treating cough, pruritus, sores tinea and snake venom (Zhang et al. 2008, Iranshahy et al. 2014). In April 2021, orange rust pustules were observed on C. acuminata, C. edulis and C. racemosa in Shaanxi Province (34°4'56'' N, 108°2'9'' E, alt. 770 m), China. Samples were collected and voucher specimens were preserved in the Herbarium Mycologicum Academiae Sinicae (nos. HMAS249947-HMAS249949), China. Consequent geospatial investigations revealed that diseased plants can be observed at an altitude of 400-1000 m, and show an incidence from 40% to 80% varied by altitude. Spermogonia epiphyllous, subcuticular, densely grouped, oval or round, 0.14-0.36 × 0.09-0.30 mm, pale orange-yellow, and type 3 of Cummins and Hiratsuka (1963). Aecia mostly hypophyllous, subepidermal without peridia, Caeoma-type, erumpent, densely grouped, oval or round, 0.27-0.85 × 0.15-0.43 mm, and orange-yellow; hyaline peridial cells produced in a periphery of the sorus under the ruptured epidermis of host plants. Aeciospores globoid or broadly ellipsoid, catenulate with intercalary cells, 15.7-20.1 × 10.8-15.7 µm, yellow to pale orange; walls hyaline, verrucose, 1.7-3.1 µm thick. This fungus was morphologically identified as Melampsora (Melampsoraceae). The rDNA-28S and the internal transcribed spacer (ITS) regions were amplified using primers NL1/NL4 and ITS1/ITS4 (Ji et al. 2020; Wang et al. 2020). Bi-directional sequences were assembled and deposited in GenBank (accession nos. MW990091-MW990093 and MW996576-MW996578). Phylogenetic trees were constructed with the ITS+rDNA-28S dataset based on maximum-likelihood (ML), maximum-parsimony (MP) and Bayesian Inference (BI). ML and MP bootstrap values were calculated by bootstrap analyses of 1,000 replicates using MEGA-X (Kumar et al. 2018), while BI posterior probabilities (Bpps) were calculated using MrBayes ver. 3.1.2 (Ji et al. 2020; Wang et al. 2020). Phylogenetic analyses grouped our specimens and Melampsora ferrinii Toome & Aime into one clade, highly supported by bootstrap values of ML, MP, and Bpps of 100%/100%/1. Inoculations were conducted with 1-year-old plants of original host, Salix babylonica L. (Toome & Aime 2015). Aeciospores suspension with a concentration of 106 spores/ml were sprayed on 20 healthy leaves, with another 20 healthy leaves sprayed with sterile water as the control. The inoculated plants were kept in darkness at 20-25 °C for 2 days and then transferred into greenhouse at 23°C with 16 h light per day. After 8-10 days of inoculation, yellow pustules of uredinia appeared on abaxial surfaces of the inoculated leaves, which were identical to Toome & Aime (2015) reported, while the control leaves remained healthy. Inoculations with the same method were conducted by spraying urediniospores, and the same rust symptoms developed after 8 days. Genus Corydalis was verified as the alternate host of M. chelidonii-pierotii Tak. Matsumoto, M. coleosporioides Dietel, M. idesiae Miyabe and M. yezoensis Miyabe & T. Matsumoto (Shinyama & Yamaoka 2012; Okane et al. 2014; Yamaoka & Okane 2019), and C. incisa (Thunb.) Pers. was speculated as the potential alternate host of M. ferrinii (Toome & Aime 2015). Based on morphology, phylogeny and pathogenicity, we firstly report M. ferrinii in mainland China and verify C. acuminata, C. edulis and C. racemosa instead of C. incisa as its alternate hosts.

Luteodorsum huanglongense (Gomphaceae, Gomphales), a New Genus and Species of Gomphoid Fungus from the Loess Plateau, Northwest China.

During an investigation of the macrofungal flora in the Huanglong Mountains of the Loess Plateau, northwest China, a unique gomphoid fungus was discovered and collected. After morphological identification and molecular phylogenetic analyses, a new genus named Luteodorsum and its type species, L. huanglongense, were proposed. Phylogenetic analyses were conducted using datasets of nuclear ribosomal DNA 28S large subunit (LSU), mitochondrial (mt) adenosine triphosphatase (ATPase) subunit 6 (atp6), and mt small-subunit rDNA (mtSSU). The results confirmed that L. huanglongense forms an independent clade within Gomphales, with full maximum likelihood bootstrap support (MLBS), maximum parsimony bootstrap support (MPBS), and Bayesian posterior probability (BPP). L. huanglongense is characterized by its sandy-brown, orange-brown, or coffee-brown color; clavate to infundibuliform shape; wrinkled and ridged hymenophore; ellipsoid to obovoid warted basidiospores; cylindrical to clavate flexuous pleurocystidia; and crystal basal mycelium. Overall, this study contributes to the growing body of knowledge on the diversity and evolution of Gomphales and provides valuable insights into the unique fungal flora found in the Huanglong Mountains.

Multinuclei Occurred Under Cryopreservation and Enhanced the Pathogenicity of Melampsora larici-populina.

Melampsora larici-populina is a macrocyclic rust, and the haploid stage with two nuclei and the diploid of mononuclear sequentially occur annually. During the preservation of dry urediniospores at -80°C, we found that one isolate, ΔTs06, was different from the usual wild-type isolate Ts06 at -20°C because it has mixed polykaryotic urediniospores. However, the other spores, including the 0, I, III, and IV stages of a life cycle, were the same as Ts06. After five generations of successive inoculation and harvest of urediniospores from the compatible host Populus purdomii, the isolate ΔTs06 steadily maintained more than 20% multiple nucleus spores. To test the pathogenesis variation of ΔTs06, an assay of host poplars was applied to evaluate the differences between ΔTs06 and Ts06. After ΔTs06 and Ts06 inoculation, leaves of P. purdomii were used to detect the expression of small secreted proteins (SSPs) and fungal biomasses using quantitative real-time PCR (qRT-PCR) and trypan blue staining. ΔTs06 displayed stronger expression of five SSPs and had a shorter latent period, a higher density of uredinia, and higher DNA mass. A transcriptomic comparison between ΔTs06 and Ts06 revealed that 3,224 were differentially expressed genes (DEGs), 55 of which were related to reactive oxygen species metabolism, the Mitogen-activated protein kinase (MAPK) signaling pathway, and the meiosis pathway. Ten genes in the mitotic and meiotic pathways and another two genes associated with the "response to DNA damage stimulus" all had an upward expression, which were detected by qRT-PCR in ΔTs06 during cryopreservation. Gas chromatography-mass spectrometry (GC-MS) confirmed that the amounts of hexadecanoic acid and octadecadienoic acid were much more in ΔTs06 than in Ts06. In addition, using spectrophotometry, hydrogen peroxide (H2O2) was also present in greater quantities in ΔTs06 compared with those found in Ts06. Increased fatty acids metabolism could prevent damage to urediniospores in super-low temperatures, but oxidant species that involved H2O2 may destroy tube proteins of mitosis and meiosis, which could cause abnormal nuclear division and lead to multinucleation, which has a different genotype. Therefore, the multinuclear isolate is different from the wild-type isolate in terms of phenotype and genotype; this multinucleation phenomenon in urediniospores improves the pathogenesis and environmental fitness of M. larici-populina.