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With >7000 species the order of rust fungi has a disproportionately large impact on agriculture, horticulture, forestry and foreign ecosystems. The infectious spores are typically dikaryotic, a feature unique to fungi in which two haploid nuclei reside in the same cell. A key example is Phakopsora pachyrhizi, the causal agent of Asian soybean rust disease, one of the world's most economically damaging agricultural diseases. Despite P. pachyrhizi's impact, the exceptional size and complexity of its genome prevented generation of an accurate genome assembly. Here, we sequence three independent P. pachyrhizi genomes and uncover a genome up to 1.25 Gb comprising two haplotypes with a transposable element (TE) content of ~93%. We study the incursion and dominant impact of these TEs on the genome and show how they have a key impact on various processes such as host range adaptation, stress responses and genetic plasticity., (© 2023. The Author(s).)

Cell surface receptors play essential roles in perceiving and processing external and internal signals at the cell surface of plants and animals. The receptor-like protein kinases (RLK) and receptor-like proteins (RLPs), two major classes of proteins with membrane receptor configuration, play a crucial role in plant development and disease defense. Although RLPs and RLKs share a similar single-pass transmembrane configuration, RLPs harbor short divergent C-terminal regions instead of the conserved kinase domain of RLKs. This RLP receptor structural design precludes sequence comparison algorithms from being used for high-throughput predictions of the RLP family in plant genomes, as has been extensively performed for RLK superfamily predictions. Here, we developed the RLPredictiOme, implemented with machine learning models in combination with Bayesian inference, capable of predicting RLP subfamilies in plant genomes. The ML models were simultaneously trained using six types of features, along with three stages to distinguish RLPs from non-RLPs (NRLPs), RLPs from RLKs, and classify new subfamilies of RLPs in plants. The ML models achieved high accuracy, precision, sensitivity, and specificity for predicting RLPs with relatively high probability ranging from 0.79 to 0.99. The prediction of the method was assessed with three datasets, two of which contained leucine-rich repeats (LRR)-RLPs from Arabidopsis and rice, and the last one consisted of the complete set of previously described Arabidopsis RLPs. In these validation tests, more than 90% of known RLPs were correctly predicted via RLPredictiOme. In addition to predicting previously characterized RLPs, RLPredictiOme uncovered new RLP subfamilies in the Arabidopsis genome. These include probable lipid transfer (PLT)-RLP, plastocyanin-like-RLP, ring finger-RLP, glycosyl-hydrolase-RLP, and glycerophosphoryldiester phosphodiesterase (GDPD, GDPDL)-RLP subfamilies, yet to be characterized. Compared to the only Arabidopsis GDPDL-RLK, molecular evolution studies confirmed that the ectodomain of GDPDL-RLPs might have undergone a purifying selection with a predominance of synonymous substitutions. Expression analyses revealed that predicted GDPGL-RLPs display a basal expression level and respond to developmental and biotic signals. The results of these biological assays indicate that these subfamily members have maintained functional domains during evolution and may play relevant roles in development and plant defense. Therefore, RLPredictiOme provides a framework for genome-wide surveys of the RLP superfamily as a foundation to rationalize functional studies of surface receptors and their relationships with different biological processes.

Machine learning (ML) is a field of artificial intelligence that has rapidly emerged in molecular biology, thus allowing the exploitation of Big Data concepts in plant genomics. In this context, the main challenges are given in terms of how to analyze massive datasets and extract new knowledge in all levels of cellular systems research. In summary, ML techniques allow complex interactions to be inferred in several biological systems. Despite its potential, ML has been underused due to complex computational algorithms and definition terms. Therefore, a systematic review to disentangle ML approaches is relevant for plant scientists and has been considered in this study. We presented the main steps for ML development (from data selection to evaluation of classification/prediction models) with a respective discussion approaching functional genomics mainly in terms of pathogen effector genes in plant immunity. Additionally, we also considered how to access public source databases under an ML framework towards advancing plant molecular biology and introduced novel powerful tools, such as deep learning., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean (Phaseolus vulgaris), and anthracnose is one of the most devastating diseases of this plant species. However, little is known about the proteins that are essential for the fungus-plant interactions. Knowledge of the fungus' arsenal of effector proteins is of great importance for understanding this pathosystem. In this work, we analyzed for the first time the arsenal of Colletotrichum lindemuthianum effector candidates (ClECs) and compared them with effector proteins from other species of the genus Colletotrichum, providing a valuable resource for studying the infection mechanisms of these pathogens in their hosts. Isolates of two physiological races (83.501 and 89 A 2 2-3) of C. lindemuthianum were used to predict 353 and 349 ClECs, respectively. Of these ClECs, 63% were found to be rich in cysteine, have repetitive sequences of amino acids, and/or possess nuclear localization sequences. Several conserved domains were found between the ClECs. We also applied the effector prediction to nine species in the genus Colletotrichum, and the results ranged from 247 predicted effectors in Colletotrichum graminicola to 446 in Colletotrichum orbiculare. Twelve conserved domains were predicted in the effector candidates of all analyzed species of Colletotrichum. An expression analysis of the eight genes encoding the effector candidates in C. lindemuthianum revealed their induction during the biotrophic phase of the fungus on the bean.

Background: Pisolithus microcarpus (Cooke & Massee) G. Cunn is a gasteromycete that produces closed basidiocarps in symbiosis with eucalypts and acacias. The fungus produces a complex basidiocarp composed of peridioles at different developmental stages and an upper layer of basidiospores free of the hyphae and ready for wind dispersal upon the rupture of the basidiocarp pellis. During basidiosporogenesis, a process that takes place inside the basidiocarp peridioles, a conspicuous reserve of fatty acids is present throughout development. While several previous studies have described basidiosporogenesis inside peridioles, very little is known about gene expression changes that may occur during this part of the fungal life cycle. The objective of this work was to analyze gene transcription during peridiole and basidiospore development, while focusing specifically on cell cycle progression and lipid metabolism., Results: Throughout different developmental stages of the peridioles we analyzed, 737 genes were regulated between adjacent compartments (>5 fold, FDR-corrected p-value < 0.05) corresponding to 3.49% of the genes present in the P. microcarpus genome. We identified three clusters among the regulated genes which showed differential expression between the peridiole developmental stages and the basidiospores. During peridiole development, transcripts for proteins involved in cellular processes, signaling, and information storage were detected, notably those for coding transcription factors, DNA polymerase subunits, DNA repair proteins, and genes involved in chromatin structure. For both internal embedded basidiospores (hereto referred to as "Internal spores", IS) and external free basidiospores (hereto referred to as "Free spores", FS), upregulated transcripts were found to involve primary metabolism, particularly fatty acid metabolism (FA). High expression of transcripts related to β-oxidation and the glyoxylate shunt indicated that fatty acids served as a major carbon source for basidiosporogenesis., Conclusion: Our results show that basidiocarp formation in P. microcarpus involves a complex array of genes that are regulated throughout peridiole development. We identified waves of transcripts with coordinated regulation and identified transcription factors which may play a role in this regulation. This is the first work to describe gene expression patterns during basidiocarp formation in an ectomycorrhizal gasteromycete fungus and sheds light on genes that may play important roles in the developmental process.

A number of genes that confer resistance to coffee leaf rust (S H 1-S H 9) have been identified within the genus Coffea, but despite many years of research on this pathosystem, the complementary avirulence genes of Hemileia vastatrix have not been reported. After identification of H. vastatrix effector candidate genes (HvECs) expressed at different stages of its lifecycle, we established an assay to characterize HvEC proteins by delivering them into coffee cells via the type-three secretion system (T3SS) of Pseudomonas syringae pv. garcae (Psgc). Employing a calmodulin-dependent adenylate cyclase assay, we demonstrate that Psgc recognizes a heterologous P. syringae T3SS secretion signal which enables us to translocate HvECs into the cytoplasm of coffee cells. Using this Psgc-adapted effector detector vector (EDV) system, we found that HvEC-016 suppresses the growth of Psgc on coffee genotypes with the S H 1 resistance gene. Suppression of bacterial blight symptoms in S H 1 plants was associated with reduced bacterial multiplication. By contrast, HvEC-016 enhanced bacterial multiplication in S H 1-lacking plants. Our findings suggest that HvEC-016 may be recognized by the plant immune system in a S H 1-dependent manner. Thus, our experimental approach is an effective tool for the characterization of effector/avirulence proteins of this important pathogen., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Asian soybean rust (ASR), caused by the fungus Phakopsora pachyrhizi, is one of the most economically important crop diseases, but is only treatable with fungicides, which are becoming less effective owing to the emergence of fungicide resistance. There are no commercial soybean cultivars with durable resistance to P. pachyrhizi, and although soybean resistance loci have been mapped, no resistance genes have been cloned. We report the cloning of a P. pachyrhizi resistance gene CcRpp1 (Cajanus cajan Resistance against Phakopsora pachyrhizi 1) from pigeonpea (Cajanus cajan) and show that CcRpp1 confers full resistance to P. pachyrhizi in soybean. Our findings show that legume species related to soybean such as pigeonpea, cowpea, common bean and others could provide a valuable and diverse pool of resistance traits for crop improvement.

Transposons are an important source of genetic variation. The phytopathogen Moniliophthora perniciosa shows high level of variability but little is known about the role of class I elements in shaping its genome. In this work, we aimed the characterization of a new gypsy/Ty3 retrotransposon species, named MpSaci, in the M. perniciosa genome. These elements are largely variable in size, ranging from 4 to 15 kb, and harbor direct long terminal repeats (LTRs) with varying degrees of similarity. Approximately, all of the copies are non-autonomous as shifts in the reading frame and stop codons were detected. Only two elements (MpSaci6 and MpSaci9) code for GAG and POL proteins that possess functional domains. Conserved domains that are typically not found in retrotransposons were detected and could potentially impact the expression of neighbor genes. Solo LTRs and several LARDs (large retrotransposon derivative) were detected. Unusual elements containing small sequences with or without interruptions that are similar to gag or different pol domains and presenting LTRs with different levels of similarities were identified. Methylation was observed in MpSaci reverse transcriptase sequences. Distribution analysis indicates that MpSaci elements are present in high copy number in the genomes of C-, S- and L-biotypes of M. perniciosa. In addition, C-biotype isolates originating from the state of Bahia have fragments in common with isolates from the Amazon region and two hybridization profiles related to two chromosomal groups. RT-PCR analysis reveals that the gag gene is constitutively expressed and that the expression is increased at least three-fold with nutrient depravation even though no new insertion were observed. These findings point out that MpSaci collaborated and, even though is primarily represented by non-autonomous elements, still might contribute to the generation of genetic variability in the most important cacao pathogen in Brazil.

Transposable elements are ubiquitous and constitute an important source of genetic variation in addition to generating deleterious mutations. Several filamentous fungi are able to defend against transposable elements using RIP(repeat-induced point mutation)-like mechanisms, which induce mutations in duplicated sequences. The sequenced Colletotrichum graminicola genome and the availability of transposable element databases provide an efficient approach for identifying and characterizing transposable elements in this fungus, which was the subject of this study. We identified 132 full-sized Tc1-Mariner transposable elements in the sequenced C. graminicola genome, which were divided into six families. Several putative transposases that have been found in these elements have conserved DDE motifs, but all are interrupted by stop codons. An in silico analysis showed evidence for RIP-generated mutations. The TCg1 element, which was cloned from the Brazilian 2908 m isolate, has a putative transposase sequence with three characteristic conserved motifs. However, this sequence is interrupted by five stop codons. Genomic DNA from various isolates was analyzed by hybridization with an internal region of TCg1. All of the isolates featured transposable elements that were similar to TCg1, and several hybridization profiles were identified. C. graminicola has many Tc1-Mariner transposable elements that have been degenerated by characteristic RIP mutations. It is unlikely that any of the characterized elements are autonomous in the sequenced isolate. The possible existence of active copies in field isolates from Brazil was shown. The TCg1 element is present in several C. graminicola isolates and is a potentially useful molecular marker for population studies of this phytopathogen., (© 2014 by The Mycological Society of America.)

Eucalypts are the world's most widely planted hardwood trees. Their outstanding diversity, adaptability and growth have made them a global renewable resource of fibre and energy. We sequenced and assembled >94% of the 640-megabase genome of Eucalyptus grandis. Of 36,376 predicted protein-coding genes, 34% occur in tandem duplications, the largest proportion thus far in plant genomes. Eucalyptus also shows the highest diversity of genes for specialized metabolites such as terpenes that act as chemical defence and provide unique pharmaceutical oils. Genome sequencing of the E. grandis sister species E. globulus and a set of inbred E. grandis tree genomes reveals dynamic genome evolution and hotspots of inbreeding depression. The E. grandis genome is the first reference for the eudicot order Myrtales and is placed here sister to the eurosids. This resource expands our understanding of the unique biology of large woody perennials and provides a powerful tool to accelerate comparative biology, breeding and biotechnology.

Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean, and the genes that encode its cell-wall-degrading enzymes are crucial for the development of the disease. Pectinases are the most important group of cell wall-degrading enzymes produced by phytopathogenic fungi. The pecC1l gene, which encodes a pectate lyase in C. lindemuthianum, was isolated and characterized. Possible cis-regulatory elements and transcription factor binding sites that may be involved in the regulation of genetic expression were detected in the promoter region of the gene. pecCl1 is represented by a single copy in the genome of C. lindemuthianum, though in silico analyses of the genomes of Colletotrichum graminicola and Colletotrichum higginsianum suggest that the genome of C. lindemuthianum includes other genes that encode pectate lyases. Phylogenetic analysis detected two groups that clustered based on different members of the pectate lyase family. Analysis of the differential expression of pecCl1 during different stages of infection showed a significant increase in pecCl1 expression five days after infection, at the onset of the necrotrophic phase. The split-maker technique proved to be an efficient method for inactivation of the pecCl1 gene, which allowed functional study of a mutant with a site-specific integration. Though gene inactivation did not result in complete loss of pectate lyase activity, the symptoms of anthracnose were reduced. Analysis of pectate lyases might not only contribute to the understanding of anthracnose in the common bean but might also lead to the discovery of an additional target for controlling anthracnose.

Boto, a class II transposable element, was characterized in the Moniliophthora perniciosa genome. The Boto transposase is highly similar to plant PIF-like transposases that belong to the newest class II superfamily known as PIF/Harbinger. Although Boto shares characteristics with PIF-like elements, other characteristics, such as the transposase intron position, the position and direction of the second ORF, and the footprint, indicate that Boto belongs to a novel family of the PIF/Harbinger superfamily. Southern blot analyses detected 6-12 copies of Boto in C-biotype isolates and a ubiquitous presence among the C- and S-biotypes, as well as a separation in the C-biotype isolates from Bahia State in Brazil in at least two genotypic groups, and a new insertion in the genome of a C-biotype isolate maintained in the laboratory for 6 years. In addition to PCR amplification from a specific insertion site, changes in the Boto hybridization profile after the M. perniciosa sexual cycle and detection of Boto transcripts gave further evidence of Boto activity. As an active family in the genome of M. perniciosa, Boto elements may contribute to genetic variability in this homothallic fungus. This is the first report of a PIF/Harbinger transposon in the genome of a phytopathogenic fungus.

Plant responses against pathogens cause up- and downward shifts in gene expression. To identify differentially expressed genes in a plant-virus interaction, susceptible tomato plants were inoculated with the potyvirus Pepper yellow mosaic virus (PepYMV) and a subtractive library was constructed from inoculated leaves at 72 h after inoculation. Several genes were identified as upregulated, including genes involved in plant defense responses (e.g., pathogenesis-related protein 5), regulation of the cell cycle (e.g., cytokinin-repressed proteins), signal transduction (e.g., CAX-interacting protein 4, SNF1 kinase), transcriptional regulators (e.g., WRKY and SCARECROW transcription factors), stress response proteins (e.g., Hsp90, DNA-J, 20S proteasome alpha subunit B, translationally controlled tumor protein), ubiquitins (e.g., polyubiquitin, ubiquitin activating enzyme 2), among others. Downregulated genes were also identified, which likewise display identity with genes involved in several metabolic pathways. Differential expression of selected genes was validated by macroarray analysis and quantitative real-time polymerase chain reaction. The possible roles played by some of these genes in the viral infection cycle are discussed.

Rust is one of the most-damaging eucalypt diseases in Brazil and is considered a potential threat to eucalypt plantations worldwide. To determine the mode of inheritance of resistance in the Eucalyptus grandis- Puccinia psidii pathosystem, ten full-sib families, generated from crosses between susceptible and resistant trees, were inoculated with a single-pustule isolate of the pathogen and rust severity was scored. The observed segregation ratios in segregating families suggested major gene control of rust resistance, although clearly incomplete penetrance, variable expressivity and minor genes are also involved in the global rust-resistance response. To identify markers linked to the resistance locus, screening of RAPD polymorphisms was conducted using bulked segregant analysis in a large full-sib family. A linkage group was built around the Ppr1 gene ( P. psidii resistance gene 1) encompassing six RAPD markers, with a genetic window spanning 5 cM with the two most-closely linked flanking markers. Besides these two flanking markers, RAPD marker AT9/917 co-segregated with Ppr1 without a single recombinant in 994 meioses. This tightly linked marker should prove useful for marker-assisted introgression and will provide an initial lead for a positional cloning effort of this resistance allele. This is the first report of a disease resistance gene identified in Eucalyptus, and one of the few examples of the involvement of a major gene in a non-coevolved pathosystem.

We used a positional cloning approach to isolate the Sw-5 disease resistance locus of tomato. Complementation experiments with overlapping cosmid clones enabled us to demonstrate that Sw-5 is a single gene locus capable of recognizing several tospovirus isolates and species. Analysis of the predicted Sw-5 protein suggests that it is a cytoplasmic protein, with a potential nucleotide binding site (NBS) domain and a C-terminal end consisting of leucine-rich repeats (LRRs). Based on its structural features, Sw-5 belongs to the class of NBS-LRR resistance genes that includes the tomato Mi, 12, and Prf genes; the Arabidopsis RPM1 gene; and the plant potato virus X resistance gene Rx. The overall similarity between the Sw-5 and Mi proteins of tomato suggests that a shared or comparable signal transduction pathway leads to both virus and nematode resistance in tomato. The similarity also supports the hypothesis that Sw-5 provides resistance via a hypersensitive response. Sw-5 is a member of a loosely clustered gene family in the telomeric region of chromosome 9. Members of this family map to other regions of chromosome 9 and also to chromosome 12, where several fungal, virus, and nematode genes have been mapped, suggesting that paralogs of Sw-5 may have evolved to provide different resistance specificities.

Cotyledon explants of tomato (Lycopersicon esculentum Mill. cvs 'Santa Clara', 'Firme' mutant, 'IPA-5' and 'IPA-6') were excised from 8- to 10-day-old in vitro-grown seedlings. Four different shoot induction media supplemented with timentin (300 mg l -1 ) were screened. When cotyledon explants were cultured on MS-based medium with 1.0 mg l -1 zeatin plus 0.1 mg l -1 IAA and supplemented with timentin, higher regeneration frequencies and a greater number of elongated shoots were obtained. It was observed that timentin caused an increase in the morphogenesis of in vitro cotyledon explants of tomato cultivars. In two of three cultivars tested, rooting of shoots was positively influenced, both in the presence and absence of timentin in the rooting medium, among shoots regenerated from explants derived from timentin-supplemented medium. The results confirm those of a previous investigation on the beneficial effects of this class of antibiotics on tomato regeneration and, consequently, its reliability for use in the transformation of this species.

Hyperhydricity in regenerated pepper plants was monitored by the induction of the ER-luminal resident protein, as observed by immunoblotting. Immunoblotting of total protein using an anti-soybean BiP serum indicated that the induction and accumulation of an 80-kDa protein was related to BiP (Binding protein), a 78-kDa ER-resident molecular chaperone. The anti-BiP serum cross-reacted with an 80-kDa protein which was significantly induced by hyperhydricity. Based on similar molecular weight and immunological reactivity we concluded that the 80-kDa protein induced in hyperhydric plants is a BiP homologue. The ultrastructural organisation of leaves in non-hyperhydric and hyperhydric pepper (Capsicum annuum L.) plants was investigated with the aim of identifying the subcellular changes associated with this phenomenon. In non-hyperhydric leaves the chloroplasts of the palisade cells had normally developed thylakoids and grana and a low accumulation or absence of starch grains and plastoglobules. In the hyperhydric plants, however, the chloroplasts exhibited thylakoid disorganisation, low grana number, an accumulation of large starch grains and a low accumulation or absence of plastoglobules. Although the structure of mitochondria and peroxisomes did not change in hyperhydric plants, the number of peroxisomes did increase.

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400 kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100 kb, spanned by nine overlapping cosmid clones.

The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.