The locoweed endophyte Alternaria oxytropis affects root development in Arabidopsis in vitro through auxin signaling and polar transport.

Locoweeds are leguminous forbs known for their toxicity to livestock caused by the endophytic fungi Alternaria sect. Undifilum. Unlike the defensive mutualisms reported in many toxin-producing endophytes and their plant hosts, the benefits that A. sect. Undifilum can confer to it host plants remains unclear. Here, we conducted physiological and genetic analyses to show that A. (sect. Undifilum) oxytropis influences growth, especially root development, in its locoweed host Oxytropis ochrocephala and Arabidopsis. The presence of A. oxytropis significantly decreased primary root length while increasing the numbers of lateral roots and root hairs, and increasing plant leaf area and fresh weight. The fungus also increased the concentrations of plant endogenous auxin, and the expression of key genes for auxin biosynthesis, signaling, and transport. These effects on root development were abolished in mutants deficient in auxin signaling and polar transport. Alternaria oxytropis down-regulated expression of PIN1 but increased expression of PIN2, PIN7, and AUX1, which might reflect alterations in the spatial accumulation of auxin responsible for the changes in root architecture. Plant growth was insensitive to A. oxytropis when naphthylphthalamic acid was applied. Our findings indicate a function of A. oxytropis in promoting the growth and development of Arabidopsis via the regulation of auxin, which in turn suggests a possible role in benefiting its locoweed hosts via a process independent of its toxin production.

Host Genotype and Precipitation Influence of Fungal Endophyte Symbiosis and Mycotoxin Abundance in a Locoweed.

Many plant endophytes produce mycotoxins, but how host genetic variation influences endophyte colonization and mycotoxin production under natural conditions is poorly understood. This interaction has not been fully considered in many previous studies which used controlled experiments with agronomic or model plant species. Here, we investigated this interaction in a naturally occurring forb (a locoweed species) Oxytropis ochrocephala, its symbiotic endophyte Alternaria oxytropis, and the mycotoxin swainsonine. Host genetic variation was characterized by microsatellite markers. Endophyte infection rate and swainsonine levels were determined by PCR and HPLC, respectively. Genetic markers defined two distinct host populations and revealed that host genetics were significantly correlated with geographical location, elevation, and precipitation. As the host diverged, symbiotic interactions were reduced or failed to produce detectable swainsonine in one host population. Host genotype and precipitation had a significant impact in shaping swainsonine production at the population level. This study highlights the effect of host genotype in influencing this interaction in locoweeds.

Alkaloids from Oxytropis ochrocephala and antiproliferative activity of sophoridine derivatives against cancer cell lines.

Ten alkaloids (1-10), with sophoridine (1) as the most abundant component, were obtained from the whole plants of Oxytropis ochrocephala Bunge. Furthermore, eight new sophoridine derivatives (11-16, 20, 21), with modification on the C-14 position of 1 were synthesized. All compounds (1-16, 20, 21) were evaluated for antiproliferative activity against five human tumor cell lines. Among them, the newly synthesized derivative 20 exhibited the best inhibitory activity against the tested cell lines. Its activity was increased by more than fourfold as compared with parent compound 1.

Ochrocephalamine A, a new quinolizidine alkaloid from Oxytropis ochrocephala Bunge.

One dimeric matrine-type alkaloid, ochrocephalamine A (1), was isolated from the poisonous plant Oxytropis ochrocephala Bunge. Its structure was elucidated by spectroscopic data and single-crystal X-ray diffraction. The insecticidal and cytotoxic activities of 1 were evaluated.

The complete chloroplast genome of Oxytropis ochrocephala Bunge 1874 (Fabaceae) and its phylogenetic analysis.

This study presents the first-ever complete chloroplast (cp) genome sequence of Oxytropis ochrocephala Bunge 1874, a member of the Fabaceae family. The cp genome spans 126,996 base pairs and includes 109 genes, comprising 76 protein-coding genes, 29 tRNA genes, and four rRNA genes. Notably, the genome lacks an inverted repeat (IR) region. Additionally, we constructed phylogenetic trees for 34 species within Trib. Galegeae, employing both maximum likelihood (ML) and Bayesian inference (BI) methods. These analyses robustly support the monophyly of the Oxytropis species, evidenced by high bootstrap values (BP = 100) and posterior probabilities (PP = 1). Within this clade, O. ochrocephala exhibits a sister relationship with other Oxytropis species. Our findings provide valuable insights into the genetic makeup and evolutionary relationships of O. ochrocephala within the Galegeae tribe.

Multiomics Reveals Mechanisms of Alternaria oxytropis Inhibiting Pathogenic Fungi in Oxytropis ochrocephala.

Endophytic fungi can benefit the host plant and increase the plant resistance. Now, there is no in-depth study of how Alternaria oxytropis (A. oxytropis) is enhancing the ability of inhibiting pathogenic fungi in Oxytropis ochrocephala (O. ochrocephala). In this study, the fungal community and metabolites associated with endophyte-infected (EI) and endophyte-free (EF) O. ochrocephala were compared by multiomics. The fungal community indicated that there was more A. oxytropis, less phylum Ascomycota, and less genera Leptosphaeria, Colletotrichum, and Comoclathris in the EI group. As metabolic biomarkers, the levels of swainsonine and apigenin-7-O-glucoside-4-O-rutinoside were significantly increased in the EI group. Through in vitro validation experiments, swainsonine and apigenin-7-O-glucoside-4-O-rutinoside can dramatically suppress the growth of pathogenic fungi Leptosphaeria sclerotioides and Colletotrichum americae-borealis by increasing the level of oxidative stress. This work suggested that O. ochrocephala containing A. oxytropis could increase the resistance to fungal diseases by markedly enhancing the content of metabolites inhibiting pathogenic fungi.

Assembly of high-quality genomes of the locoweed Oxytropis ochrocephala and its endophyte Alternaria oxytropis provides new evidence for their symbiotic relationship and swainsonine biosynthesis.

Locoweeds are perennial forbs poisonous to livestock and cause extreme losses to animal husbandry. Locoweed toxicity is attributed to the symbiotic endophytes in Alternaria sect. Undifilum, which produce a mycotoxin swainsonine (SW). We performed a de novo whole genome sequencing of the most common locoweed in China, Oxytropis ochrocephala (2n = 16), and assembled a high-quality, chromosome-level reference genome. Its genome size is 958.83 Mb with 930.94 Mb (97.09%) anchored and oriented onto eight chromosomes, and 31,700 protein-coding genes were annotated. Phylogenetic and collinearity analysis showed it is closely related to Medicago truncatula with a pair of large interchromosomal rearrangements, and both species underwent a whole-genome duplication event. We also derived the genome of A. oxytropis at 74.48 Mb with a contig N50 of 8.87 Mb and 10,657 protein-coding genes, and refined the genes of SW biosynthesis. Multiple Alternaria species containing the swnK gene were grouped into a single clade, but in other genera, swnK's homologues are diverse. Resequencing of 41 A. oxytropis strains revealed one SNP in the SWN cluster causing changes in SW concentration. Comparing the transcriptomes of symbiotic and nonsymbiotic interactions identified differentially expressed genes (DEGs) linked to defence and secondary metabolism in the host. Within the endophyte DEGs were linked to cell wall degradation, fatty acids and nitrogen metabolism. Symbiosis induced the upregulation of most of the SW biosynthetic genes. These two genomes and relevant sequencing data should provide valuable genetic resources for the study of the evolution, interaction, and SW biosynthesis in the symbiont.

De novo Transcriptome Assembly of a Chinese Locoweed (Oxytropis ochrocephala) Species Provides Insights into Genes Associated with Drought, Salinity, and Cold Tolerance.

BACKGROUND: Locoweeds (toxic Oxytropis and Astraglus species), containing the toxic agent swainsonine, pose serious threats to animal husbandry on grasslands in both China and the US. Some locoweeds have evolved adaptations in order to resist various stress conditions such as drought, salt and cold. As a result they replace other plants in their communities and become an ecological problem. Currently very limited genetic information of locoweeds is available and this hinders our understanding in the molecular basis of their environmental plasticity, and the interaction between locoweeds and their symbiotic swainsonine producing endophytes. Next-generation sequencing provides a means of obtaining transcriptomic sequences in a timely manner, which is particularly useful for non-model plants. In this study, we performed transcriptome sequencing of Oxytropis ochrocephala plants followed by a de nove assembly. Our primary aim was to provide an enriched pool of genetic sequences of an Oxytropis sp. for further locoweed research. RESULTS: Transcriptomes of four different O. ochrocephala samples, from control (CK) plants, and those that had experienced either drought (20% PEG), salt (150 mM NaCl) or cold (4°C) stress were sequenced using an Illumina Hiseq 2000 platform. From 232,209,506 clean reads 23,220,950,600 (~23 G nucleotides), 182,430 transcripts and 88,942 unigenes were retrieved, with an N50 value of 1237. Differential expression analysis revealed putative genes encoding heat shock proteins (HSPs) and late embryogenesis abundant (LEA) proteins, enzymes in secondary metabolite and plant hormone biosyntheses, and transcription factors which are involved in stress tolerance in O. ochrocephala. In order to validate our sequencing results, we further analyzed the expression profiles of nine genes by quantitative real-time PCR. Finally, we discuss the possible mechanism of O. ochrocephala's adaptations to stress environment. CONCLUSION: Our transcriptome sequencing data present useful genetic information of a locoweed species. This genetic information will underpin further research in elucidating the environmental acclimation mechanism in locoweeds and the endophyte-plant association.

Selection of appropriate reference genes for quantitative real-time PCR in Oxytropis ochrocephala Bunge using transcriptome datasets under abiotic stress treatments.

BACKGROUND: Oxytropis ochrocephala Bunge, an indigenous locoweed species in China, poses great threats to livestock on grasslands. There is a need for further genetic study in the plants per se, for understanding the basis of its acclimation mechanism in various unfavorable environmental conditions and to implement effective control measures. Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method for gene expression analysis. To facilitate gene expression studies and obtain more accurate qRT-PCR data, normalization relative to stable reference genes is required. The aim of this study was to select the most stable reference genes for transcriptional analysis in O. ochrocephala. RESULTS: We selected 12 candidate reference genes, 18S ribosomal RNA (18S RNA), actin2/7 (ACT7), ß-actin (ACTB), actin101 (ACT101), actin11 (ACT11), ß-tubulin (TUB), α-tubulin (TUA), glyceraldehyde-3-phosphate dehydrogenase-1 (GAPDH1), GAPDH2, metallothionein-like protein (MET), fructose-bisphosphate aldolase (FBA) and histone H3 (HIS), from the transcriptome datasets of O. ochrocephala and determined the suitability by analyzing their expression levels when exposed to a range of abiotic stress conditions. By employing software packages including geNorm, NormFinder and BestKeeper, HIS, ACT7, and ACT101 were assessed as the most suitable set for normalization in all samples. When normalized with the most stable reference genes, the expression patterns of the three target genes were in accordance with those in the transcriptome data, indicating that the reference genes selected in this study are suitable. CONCLUSIONS: The study provided appropriate reference genes for accurate normalization in qRT-PCR analysis in O. ochrocephala and emphasized the importance of validating reference genes for gene expression analysis under specific experimental condition. The usage of inappropriate reference gene would cause misinterpretation.