AGRAMP: machine learning models for predicting antimicrobial peptides against phytopathogenic bacteria.

Introduction: Antimicrobial peptides (AMPs) are promising alternatives to traditional antibiotics for combating plant pathogenic bacteria in agriculture and the environment. However, identifying potent AMPs through laborious experimental assays is resource-intensive and time-consuming. To address these limitations, this study presents a bioinformatics approach utilizing machine learning models for predicting and selecting AMPs active against plant pathogenic bacteria. Methods: N-gram representations of peptide sequences with 3-letter and 9-letter reduced amino acid alphabets were used to capture the sequence patterns and motifs that contribute to the antimicrobial activity of AMPs. A 5-fold cross-validation technique was used to train the machine learning models and to evaluate their predictive accuracy and robustness. Results: The models were applied to predict putative AMPs encoded by intergenic regions and small open reading frames (ORFs) of the citrus genome. Approximately 7% of the 10,000-peptide dataset from the intergenic region and 7% of the 685,924-peptide dataset from the whole genome were predicted as probable AMPs. The prediction accuracy of the reported models range from 0.72 to 0.91. A subset of the predicted AMPs was selected for experimental test against Spiroplasma citri, the causative agent of citrus stubborn disease. The experimental results confirm the antimicrobial activity of the selected AMPs against the target bacterium, demonstrating the predictive capability of the machine learning models. Discussion: Hydrophobic amino acid residues and positively charged amino acid residues are among the key features in predicting AMPs by the Random Forest Algorithm. Aggregation propensity appears to be correlated with the effectiveness of the AMPs. The described models would contribute to the development of effective AMP-based strategies for plant disease management in agricultural and environmental settings. To facilitate broader accessibility, our model is publicly available on the AGRAMP (Agricultural Ngrams Antimicrobial Peptides) server.

Cacao pod transcriptome profiling of seven genotypes identifies features associated with post-penetration resistance to Phytophthora palmivora.

The oomycete Phytophthora palmivora infects the fruit of cacao trees (Theobroma cacao) causing black pod rot and reducing yields. Cacao genotypes vary in their resistance levels to P. palmivora, yet our understanding of how cacao fruit respond to the pathogen at the molecular level during disease establishment is limited. To address this issue, disease development and RNA-Seq studies were conducted on pods of seven cacao genotypes (ICS1, WFT, Gu133, Spa9, CCN51, Sca6 and Pound7) to better understand their reactions to the post-penetration stage of P. palmivora infection. The pod tissue-P. palmivora pathogen assay resulted in the genotypes being classified as susceptible (ICS1, WFT, Gu133 and Spa9) or resistant (CCN51, Sca6 and Pound7). The number of differentially expressed genes (DEGs) ranged from 1625 to 6957 depending on genotype. A custom gene correlation approach identified 34 correlation groups. De novo motif analysis was conducted on upstream promoter sequences of differentially expressed genes, identifying 76 novel motifs, 31 of which were over-represented in the upstream sequences of correlation groups and associated with gene ontology terms related to oxidative stress response, defense against fungal pathogens, general metabolism and cell function. Genes in one correlation group (Group 6) were strongly induced in all genotypes and enriched in genes annotated with defense-responsive terms. Expression pattern profiling revealed that genes in Group 6 were induced to higher levels in the resistant genotypes. An additional analysis allowed the identification of 17 candidate cis-regulatory modules likely to be involved in cacao defense against P. palmivora. This study is a comprehensive exploration of the cacao pod transcriptional response to P. palmivora spread after infection. We identified cacao genes, promoter motifs, and promoter motif combinations associated with post-penetration resistance to P. palmivora in cacao pods and provide this information as a resource to support future and ongoing efforts to breed P. palmivora-resistant cacao.

Draft genome sequence of Xylella fastidiosa strain ATCC 35874 isolated from infected red oak in Washington, DC.

We report here the draft genome sequence of Xylella fastidiosa strain ATCC 35874. The strain was originally isolated from infected red oak in Washington, DC, and obtained from the American Type Culture Collection. The ATCC 35874 genome contains 2,543,332 bp and has a G + C content of 51.72%.

Composition of the alfalfa pathobiome in commercial fields.

Through the recent advances of modern high-throughput sequencing technologies, the "one microbe, one disease" dogma is being gradually replaced with the principle of the "pathobiome". Pathobiome is a comprehensive biotic environment that not only includes a diverse community of all disease-causing organisms within the plant but also defines their mutual interactions and resultant effect on plant health. To date, the concept of pathobiome as a major component in plant health and sustainable production of alfalfa (Medicago sativa L.), the most extensively cultivated forage legume in the world, is non-existent. Here, we approached this subject by characterizing the biodiversity of the alfalfa pathobiome using high-throughput sequencing technology. Our metagenomic study revealed a remarkable abundance of different pathogenic communities associated with alfalfa in the natural ecosystem. Profiling the alfalfa pathobiome is a starting point to assess known and identify new and emerging stress challenges in the context of plant disease management. In addition, it allows us to address the complexity of microbial interactions within the plant host and their impact on the development and evolution of pathogenesis.

Genome Resources for the Colletotrichum gloeosporioides Species Complex: 13 Tree Endophytes from the Neotropics and Paleotropics.

Thirteen draft genome assemblies are presented for four Colletotrichum gloeosporioides complex species, namely, Colletotrichum aeschynomenes, Colletotrichum asianum, Colletotrichum fructicola, and Colletotrichum siamense, which were isolated from tropical tree hosts as endophytes.

Comprehensive profiling of endogenous phytohormones and expression analysis of 1-aminocyclopropane-1-carboxylic acid synthase gene family during fruit development and ripening in octoploid strawberry (Fragaria× ananassa).

The non-climacteric octoploid strawberry (Fragaria × ananassa Duchesne ex Rozier) was used as a model to study its regulation during fruit ripening. High performance liquid chromatography electrospray tandem-mass spectrometry (HPLC-ESI-MS/MS) was employed to profile 28 different endogenous phytohormones in strawberry. These include auxins, cytokinins (CKs), abscisic acid (ABA), ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC), jasmonates, and phenolic compounds salicylic acid (SA), benzoic acid (BzA) and phenylacetic acid (PAA) together with their various metabolic forms that have remained largely unexplored thus far. ABA, ACC and CK N6-(Δ2-isopentenyl)adenine (iP) were found to be associated with ripening while ABA catabolites 9-hydroxy-ABA and phaseic acid mimicked the pattern of climacteric decline at the turning phase of strawberry ripening. The content of other CK forms except iP decreased as fruit ripened, as also that of auxins indole-3-acetic acid (IAA) and oxo-IAA, and of jasmonates. Data presented here also suggest that both the transition and progression of strawberry fruit ripening are associated with N6-(Δ2-isopentenyl)adenosine-5'-monophosphate (iPRMP) → N6-(Δ2-isopentenyl)adenosine (iPR) → iP as the preferred CK metabolic pathway. In contrast, the ethylene precursor ACC was present at higher levels, with its abundance increasing from the onset of ripening to the red ripe stage. Further investigation of ripening-specific ACC accumulation revealed the presence of a large ACC synthase (ACS) encoding gene family in octoploid strawberry that was previously unknown. Seventeen ACS genes were found differentially expressed in fruit tissues, while six of them showed induced expression during strawberry fruit ripening. These data suggest a possible role(s) of ACC, ABA, and iP in strawberry fruit ripening. These data add new dimension to the existing knowledge of the interplay of different endogenous phytohormones in octoploid strawberry, paving the way for further investigation of their individual role(s) in fruit ripening.

Guilt by Association: DNA Barcoding-Based Identification of Potential Plant Hosts of Phytoplasmas from Their Insect Carriers.

Phytoplasmas are small phloem-restricted and insect-transmissible bacteria that infect many plant species, including important crops and ornamental plants, causing severe economic losses. Our previous studies screened phytoplasmas in hundreds of leafhoppers collected from natural habitats worldwide and identified multiple genetically different phytoplasmas in seven leafhopper species (potential insect vectors). As an initial step toward determining the impact of these phytoplasmas on the ecosystem, ribulose 1,5-biphosphate carboxylase large subunit (rbcL), a commonly used plant DNA barcoding marker, was employed to identify the plant species that the phytoplasma-harboring leafhoppers feed on. The DNA of 17 individual leafhoppers was PCR amplified using universal rbcL primers. PCR products were cloned, and five clones per amplicon were randomly chosen for Sanger sequencing. Moreover, Illumina high-throughput sequencing on selected PCR products was conducted and confirmed no missing targets in Sanger sequencing. The nucleotide BLAST results revealed 14 plant species, including six well-known plant hosts of phytoplasmas such as tomato, alfalfa, and maize. The remaining species have not been documented as phytoplasma hosts, expanding our knowledge of potential plant hosts. Notably, the DNA of tomato and maize (apparently cultivated in well-managed croplands) was detected in some phytoplasma-harboring leafhopper species sampled in non-crop lands, suggesting the spillover/spillback risk of phytoplasma strains between crop and non-crop areas. Furthermore, our results indicate that barcoding (or metabarcoding) is a valuable tool to study the three-way interactions among phytoplasmas, plant hosts, and vectors. The findings contribute to a better understanding of phytoplasma host range, host shift, and disease epidemiology.

Draft Genome Sequence of a Xylella fastidiosa Strain Causing Bacterial Leaf Scorch of American Elm in Washington, DC.

Here, we report the draft genome sequence of Xylella fastidiosa strain ATCC 35873, which was obtained from the American Type Culture Collection and was originally isolated from a symptomatic American elm tree grown in Washington, DC. The ATCC 35873 genome contains 2,454,216 bp and has a GC content of 51.68%.

Draft Genome Sequence of the Sophorolipid-Producing Yeast Pseudohyphozyma bogoriensis ATCC 18809.

Pseudohyphozyma bogoriensis is gaining attention as a microbial source of high-value sophorolipids. We report here on its genomic sequence, which will improve our understanding of its metabolic pathways and allow the development of genome manipulation systems. PacBio sequencing was performed, yielding a 26-Mbp genome with 57% GC content and encoding 7,847 predicted proteins.

Elucidation of tropane alkaloid biosynthesis in Erythroxylum coca using a microbial pathway discovery platform.

Tropane alkaloids (TAs) are heterocyclic nitrogenous metabolites found across seven orders of angiosperms, including Malpighiales (Erythroxylaceae) and Solanales (Solanaceae). Despite the well-established euphorigenic properties of Erythroxylaceae TAs like cocaine, their biosynthetic pathway remains incomplete. Using yeast as a screening platform, we identified and characterized the missing steps of TA biosynthesis in Erythroxylum coca. We first characterize putative E. coca polyamine synthase- and amine oxidase-like enzymes in vitro, in yeast, and in planta to show that the first tropane ring closure in Erythroxylaceae occurs via bifunctional spermidine synthase/N-methyltransferases and both flavin- and copper-dependent amine oxidases. We next identify a SABATH family methyltransferase responsible for the 2-carbomethoxy moiety characteristic of Erythroxylaceae TAs and demonstrate that its coexpression with methylecgonone reductase in yeast engineered to express the Solanaceae TA pathway enables the production of a hybrid TA with structural features of both lineages. Finally, we use clustering analysis of Erythroxylum transcriptome datasets to discover a cytochrome P450 of the CYP81A family responsible for the second tropane ring closure in Erythroxylaceae, and demonstrate the function of the core coca TA pathway in vivo via reconstruction and de novo biosynthesis of methylecgonine in yeast. Collectively, our results provide strong evidence that TA biosynthesis in Erythroxylaceae and Solanaceae is polyphyletic and that independent recruitment of unique biosynthetic mechanisms and enzyme classes occurred at nearly every step in the evolution of this pathway.

Correction: Solano-Aguilar et al. Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention. Nutrients 2021, 13, 4350.

There was an error in the original publication [...].

Delayed access to feed early post-hatch affects the development and maturation of gastrointestinal tract microbiota in broiler chickens.

BACKGROUND: The first two weeks of post-hatch (PH) growth in broilers (meat-type birds) are critical for gut development and microbiota colonization. In the current broiler production system, chicks may not receive feed and water for 24 to 72 h due to variations in hatching time and hatchery management. Post-hatch feed delay affects body weight, feed efficiency, mortality, and gut development. The goal of this study was to investigate changes in the microbiome in broiler chickens early PH and the effect of delayed access to feed on the microbiota. RESULTS: Chicks either received feed and water immediately after hatch or access to feed was delayed for 48 h to mimic commercial hatchery settings (treatment, TRT). Both groups were sampled (n = 6) at -48, 0, 4 h, and 1 (24 h), 2 (48 h), 3 (72 h), 4 (96 h), 6 (144 h), 8 (192 h), 10 (240 h), 12 (288 h) and 14 (336 h) days PH. Ileal (IL) and cecal (CE) epithelial scrapings (mucosal bacteria, M) and digesta (luminal bacteria, L) were collected for microbiota analysis. Microbiota was determined by sequencing the V3-V4 region of bacterial 16S rRNA and analyzed using QIIME2. The microbiota of early ileal and cecal samples were characterized by high abundance of unclassified bacteria. Among four bacterial populations (IL-L, IL-M, CE-L, CE-M), IL-M was the least affected by delayed access to feed early PH. Both alpha and beta diversities were affected by delayed access to feed PH in IL-L, CE-M and CE-L. However, the development effect was more pronounced. In all four bacterial populations, significant changes due to developmental effect (time relative to hatch) was observed in taxonomic composition, with transient changes of bacterial taxa during the first two weeks PH. Delayed access to feed has limited influence on bacterial composition with only a few genera and species affected in all four bacterial populations. Predicted function based on 16S rRNA was also affected by delayed access to feed PH with most changes in metabolic pathway richness observed in IL-L, CE-L and CE-M. CONCLUSIONS: These results show transient changes in chicken microbiota biodiversity during the first two weeks PH and indicate that delayed access to feed affects microbiota development. Proper microbiota development could be an important factor in disease prevention and antibiotic use in broiler chickens. Moreover, significant differences in response to delayed access to feed PH between luminal and mucosal bacterial populations strongly suggests the need for separate analysis of these two populations.

Research Note: Choice of microbiota database affects data analysis and interpretation in chicken cecal microbiota.

The chicken microbiota is often analyzed to address questions about the effects of diet or disease on poultry health. To analyze the microbiota, bioinformatic platforms such as QIIME 2 and mothur are used, which incorporate public taxonomic databases such as Greengenes, the ribosomal database project (RDP), and SILVA to assign taxonomies to bacterial sequences. Many chicken microbiota studies continue to incorporate the Greengenes database, which has not been updated since 2013. To determine whether a choice of database could affect results, this study compared the results of bioinformatic analyses obtained using the Greengenes, RDP, and SILVA databases on a cecal luminal microbiome dataset. The QIIME 2 platform was used to process 16S bacterial sequences and assign taxonomies with Greengenes, RDP, and SILVA. Linear discriminant analysis effect size (LEfSe) was performed, allowing for the comparison of taxonomies considered significantly differentially abundant between the three databases. Some notable differences between databases were observed in results, in particular the ability of SILVA database to classify members of the family Lachnospiraceae into separate genera, while these members remained in one group of unclassified Lachnospiraceae through Greengenes and RDP. LEfSe analyses showed that the SILVA database produced more differentially abundant genera, in large part due to the classification of these separate Lachnospiraceae genera. Additionally, the relative abundance of unclassified Lachnospiraceae in SILVA results was significantly lower than in RDP results. Our results show the choice of taxonomic database can influence the results of a microbiota study at the genus level, potentially affecting the interpretation of the results. The use of the SILVA database is recommended over Greengenes in chicken microbiota studies, as more specific classifications at the genus level may provide more accurate interpretations of changes in the microbiota.

Diversity of the virome associated with alfalfa (Medicago sativa L.) in the U.S. Pacific Northwest.

Alfalfa (Medicago sativa L.) is one of the most extensively cultivated forage legumes in the world. It is currently the third most valuable field crop in the United States with an estimated value of over $9.3 billion. Alfalfa productivity is limited by various infectious diseases that can reduce forage yield and quality and shorten stand life. The crop can frequently be infected with a diverse array of pathogens and other organisms that have distinct life cycles, biology, and mode of action. Among them are many coinfecting viruses, that greatly contribute to the heterogeneity of within-host pathogenic communities, representing a ubiquitous and abundant background for all other host-pathogen interactions. Regrettably, the impact of viral diseases, their role in alfalfa health and involvement in the severity of multi-pathogen infections are often underestimated and not well understood. As high-throughput sequencing approaches have been developed, opportunities to delve into these complex interactions can be realized. In this work, we have characterized a diversity of viral populations in several commercial alfalfa production fields located in the U.S. Pacific Northwest. At least 45 distinct viruses have been identified in all alfalfa samples. Among them some were known to infect the crop prior to this study, and others were designated as emerging, novel and viruses integrated into the alfalfa genome. Known viruses included alfalfa mosaic virus, pea streak virus and bean leafroll virus, while among emerging and novel agents were alfalfa virus S, cherry virus Trakiya, several rhabdoviruses and others. Additional biological and impact studies will be needed to determine if newly identified viruses, especially those that have not been reported from alfalfa before, should be considered pathogens of this crop.

Effects of Eimeria tenella on Cecal Luminal and Mucosal Microbiota in Broiler Chickens.

The intestinal disease coccidiosis, caused by parasitic Eimeria species, severely impacts poultry production, leading to an estimated $14 billion in annual losses worldwide. As the poultry industry moves away from antibiotics as a treatment for diseases, a better understanding of the microbiota is required to develop other solutions such as probiotics, prebiotics, and nutritional supplements. This study aimed to investigate the effects of Eimeria tenella infection on luminal (cecal contents [CeC]) and mucosal (cecal epithelial scrapings [CeS]) microbial populations in 288 Ross 708 broiler chickens at multiple time points postinfection (PI). By use of 16S rRNA amplicon sequencing, it was revealed that microbial diversity differed in infected (IF) chickens in comparison to the control (C) in both CeC and CeS microbiota at the peak of infection (7 days PI), when simultaneously IF birds saw reduced body weight gain and a higher feed conversion ratio. Infection resulted in a significant differential abundance of some bacterial taxa, including increases in potential secondary pathogens Escherichia coli, Enterococcus, Clostridium, and Proteus and a decrease in the short chain fatty acid-producing family Lachnospiraceae. Predicted metagenomic pathways associated with E. coli, such as those responsible for amino acid biosynthesis, were differentially expressed in IF birds. In conclusion, our results show that E. tenella infection disturbs luminal and mucosal microbiota balance in chickens. Moreover, the luminal microbiota seems to be more susceptible to prolonged imbalance due to IF, whereas the mucosal microbiota appeared to be affected only in the short term, demonstrating the importance of researching both the luminal and mucosal microbiota of the cecum.

Efectos de Eimeria tenella sobre la microbiota luminal y de la mucosa de los ciegos en pollos de engorde. La coccidiosis, una enfermedad intestinal causada por especies parasitarias de Eimeria, afecta gravemente la producción avícola, lo que genera pérdidas anuales estimadas en 14,000 millones de dólares en todo el mundo. A medida que la industria avícola se aleja de los antibióticos como tratamiento para enfermedades, se requiere de un mejor conocimiento de la microbiota para desarrollar otras soluciones como probióticos, prebióticos y suplementos nutricionales. Este estudio tuvo como objetivo investigar los efectos de la infección por Eimeria tenella en las poblaciones microbianas luminales (contenido cecal [CeC]) y de la mucosa (raspados del epitelio cecal [CeS]) en pollos de engorde Ross 708 (288) en diferentes puntos de tiempo después de la infección (PI). Mediante el uso de la secuenciación de amplicones de ARNr 16S, se reveló que la diversidad microbiana difería en los pollos infectados (IF) en comparación con el grupo control (C) tanto en la microbiota del contenido cecal como de la mucosa durante el pico de infección (7 días después de la infección), cuando de manera simultánea las aves infectadas mostraron una reducción en la ganancia de peso corporal reducido y una tasa de conversión alimenticia más alta. La infección resultó en una abundancia diferencial significativa de algunos taxones bacterianos, incluidos aumentos en los patógenos secundarios potenciales como Escherichia coli, Enterococcus, Clostridium y Proteus y una disminución en la familia Lachnospiraceae productora de ácidos grasos de cadena corta. Las vías metagenómicas predichas asociadas con E. coli, como las responsables de la biosíntesis de aminoácidos, se expresaron diferencialmente en las aves infectadas. En conclusión, estos resultados muestran que la infección por E. tenella perturba el equilibrio de la microbiota luminal y de la mucosa en pollos. Además, la microbiota luminal parece ser más susceptible a un desequilibrio prolongado debido a la infección, mientras que la microbiota mucosa parece verse afectada solo a corto plazo, lo que demuestra la importancia de investigar tanto la microbiota luminal como la de la mucosa en el ciego.

Fruit and Vegetable Supplemented Diet Modulates the Pig Transcriptome and Microbiome after a Two-Week Feeding Intervention.

A study was conducted to determine the effects of a diet supplemented with fruits and vegetables (FV) on the host whole blood cell (WBC) transcriptome and the composition and function of the intestinal microbiome. Nine six-week-old pigs were fed a pig grower diet alone or supplemented with lyophilized FV equivalent to half the daily recommended amount prescribed for humans by the Dietary Guideline for Americans (DGA) for two weeks. Host transcriptome changes in the WBC were evaluated by RNA sequencing. Isolated DNA from the fecal microbiome was used for 16S rDNA taxonomic analysis and prediction of metabolomic function. Feeding an FV-supplemented diet to pigs induced differential expression of several genes associated with an increase in B-cell development and differentiation and the regulation of cellular movement, inflammatory response, and cell-to-cell signaling. Linear discriminant analysis effect size (LEfSe) in fecal microbiome samples showed differential increases in genera from Lachnospiraceae and Ruminococcaceae families within the order Clostridiales and Erysipelotrichaceae family with a predicted reduction in rgpE-glucosyltransferase protein associated with lipopolysaccharide biosynthesis in pigs fed the FV-supplemented diet. These results suggest that feeding an FV-supplemented diet for two weeks modulated markers of cellular inflammatory and immune function in the WBC transcriptome and the composition of the intestinal microbiome by increasing the abundance of bacterial taxa that have been associated with improved intestinal health.

Targeted transcriptomics reveals signatures of large-scale independent origins and concerted regulation of effector genes in Radopholus similis.

The burrowing nematode, Radopholus similis, is an economically important plant-parasitic nematode that inflicts damage and yield loss to a wide range of crops. This migratory endoparasite is widely distributed in warmer regions and causes extensive destruction to the root systems of important food crops (e.g., citrus, banana). Despite the economic importance of this nematode, little is known about the repertoire of effectors owned by this species. Here we combined spatially and temporally resolved next-generation sequencing datasets of R. similis to select a list of candidates for the identification of effector genes for this species. We confirmed spatial expression of transcripts of 30 new candidate effectors within the esophageal glands of R. similis by in situ hybridization, revealing a large number of pioneer genes specific to this nematode. We identify a gland promoter motif specifically associated with the subventral glands (named Rs-SUG box), a putative hallmark of spatial and concerted regulation of these effectors. Nematode transcriptome analyses confirmed the expression of these effectors during the interaction with the host, with a large number of pioneer genes being especially abundant. Our data revealed that R. similis holds a diverse and emergent repertoire of effectors, which has been shaped by various evolutionary events, including neofunctionalization, horizontal gene transfer, and possibly by de novo gene birth. In addition, we also report the first GH62 gene so far discovered for any metazoan and putatively acquired by lateral gene transfer from a bacterial donor. Considering the economic damage caused by R. similis, this information provides valuable data to elucidate the mode of parasitism of this nematode.

Mitochondrial Genomics of Six Cacao Pathogens From the Basidiomycete Family Marasmiaceae.

Thread blight disease has recently been described as an emerging disease on cacao (Theobroma cacao) in Ghana. In Ghana, thread blight disease is caused by multiple species of the Marasmiaceae family: Marasmius tenuissimus, M. crinis-equi, M. palmivorus, and Marasmiellus scandens. Interestingly, two additional members of the Marasmiaceae; Moniliophthora roreri (frosty pod rot) and Moniliophthora perniciosa (witches' broom disease), are major pathogens of cacao in the Western hemisphere. It is important to accurately characterize the genetic relationships among these economically important species in support of their disease management. We used data from Illumina NGS-based genome sequencing efforts to study the mitochondrial genomes (mitogenomes) of the four cacao thread blight associated pathogens from Ghana and compared them with published mitogenomes of Mon. roreri and Mon. perniciosa. There is a remarkable interspecies variation in mitogenome size within the six cacao-associated Marasmiaceae species, ranging from 43,121 to 109,103 bp. The differences in genome lengths are primarily due to the number and lengths of introns, differences in intergenic space, and differences in the size and numbers of unidentified ORFs (uORF). Among seven M. tenuissimus mitogenomes sequenced, there is variation in size and sequence pointing to divergent evolution patterns within the species. The intronic regions show a high degree of sequence variation compared to the conserved sequences of the 14 core genes. The intronic ORFs identified, regardless of species, encode GIY-YIG or LAGLIDADG domain-containing homing endonuclease genes. Phylogenetic relationships using the 14 core proteins largely mimic the phylogenetic relationships observed in gene order patterns, grouping M. tenuissimus with M. crinis-equi, and M. palmivorus with Mon. roreri and Mon. perniciosa, leaving Mar. scandens as an outlier. The results from this study provide evidence of independent expansion/contraction events and sequence diversification in each species and establish a foundation for further exploration of the evolutionary trajectory of the fungi in Marasmiaceae family.

Genomic analysis of the polyamine biosynthesis pathway in duckweed Spirodela polyrhiza L.: presence of the arginine decarboxylase pathway, absence of the ornithine decarboxylase pathway, and response to abiotic stresses.

MAIN CONCLUSION: Identification of the polyamine biosynthetic pathway genes in duckweed S. polyrhiza reveals presence of prokaryotic as well as land plant-type ADC pathway but absence of ODC encoding genes. Their differential gene expression and transcript abundance is shown modulated by exogenous methyl jasmonate, salinity, and acidic pH. Genetic components encoding for polyamine (PA) biosynthetic pathway are known in several land plant species; however, little is known about them in aquatic plants. We utilized recently sequenced three duckweed (Spirodela polyrhiza) genome assemblies to map PA biosynthetic pathway genes in S. polyrhiza. PA biosynthesis in most higher plants except for Arabidopsis involves two pathways, via arginine decarboxylase (ADC) and ornithine decarboxylase (ODC). ADC-mediated PA biosynthetic pathway genes, namely, one arginase (SpARG1), two arginine decarboxylases (SpADC1, SpADC2), one agmatine iminohydrolase/deiminase (SpAIH), one N-carbamoyl putrescine amidase (SpCPA), three S-adenosylmethionine decarboxylases (SpSAMDc1, 2, 3), one spermidine synthase (SpSPDS1) and one spermine synthase (SpSPMS1) in S. polyrhiza genome were identified here. However, no locus was found for ODC pathway genes in this duckweed. Hidden Markov Model protein domain analysis established that SpADC1 is a prokaryotic/biodegradative type ADC and its molecular phylogenic classification fell in a separate prokaryotic origin ADC clade with SpADC2 as a biosynthetic type of arginine decarboxylase. However, thermospermine synthase (t-SPMS)/Aculis5 genes were not found present. Instead, one of the annotated SPDS may also function as SPMS, since it was found associated with the SPMS phylogenetic clade along with known SPMS genes. Moreover, we demonstrate that S. polyrhiza PA biosynthetic gene transcripts are differentially expressed in response to unfavorable conditions, such as exogenously added salt, methyl jasmonate, or acidic pH environment as well as in extreme temperature regimes. Thus, S. polyrhiza genome encodes for complete polyamine biosynthesis pathway and the genes are transcriptionally active in response to changing environmental conditions suggesting an important role of polyamines in this aquatic plant.