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Background RNA sequencing allows the measuring of gene expression at a resolution unmet by expression arrays or RT-qPCR. It is however necessary to normalize sequencing data by library size, transcript size and composition, among other factors, before comparing expression levels. The use of internal control genes or spike-ins is advocated in the literature for scaling read counts, but the methods for choosing reference genes are mostly targeted at RT-qPCR studies and require a set of pre-selected candidate controls or pre-selected target genes. Results Here, we report an R-based pipeline to select internal control genes based solely on read counts and gene sizes. This novel method first normalizes the read counts to Transcripts per Million (TPM) and then excludes weakly expressed genes using the DAFS script to calculate the cut-off. It then selects as references the genes with lowest TPM coefficient of variation. We used this method to pick custom reference genes for the differential expression analysis of three transcriptome sets from transgenic Arabidopsis plants expressing heterologous fungal effector proteins tagged with GFP (using GFP alone as the control). The custom reference genes showed lower coefficient of variation and fold change as well as a broader range of expression levels than commonly used reference genes. When analyzed with NormFinder, both typical and custom reference genes were considered suitable internal controls, but the custom selected genes were more stably expressed. geNorm produced a similar result in which most custom selected genes ranked higher (i.e. were more stably expressed) than commonly used reference genes. Conclusions The proposed method is innovative, rapid and simple. Since it does not depend on genome annotation, it can be used with any organism, and does not require pre-selected reference candidates or target genes that are not always available.

Contents Summary 1190 I. Introduction 1190 II. Rust fungi: a diverse and serious threat to agriculture 1191 III. The different facets of rust life cycles and unresolved questions about their evolution 1191 IV. The biology of rust infection 1192 V. Rusts in the genomics era: the ever-expanding list of candidate effector genes 1195 VI. Functional characterization of rust effectors 1197 VII. Putting rusts to sleep: Pucciniales research outlooks 1201 Acknowledgements 1202 References 1202 SUMMARY: Rust fungi (Pucciniales) are the largest group of plant pathogens and represent one of the most devastating threats to agricultural crops worldwide. Despite the economic importance of these highly specialized pathogens, many aspects of their biology remain obscure, largely because rust fungi are obligate biotrophs. The rise of genomics and advances in high-throughput sequencing technology have presented new options for identifying candidate effector genes involved in pathogenicity mechanisms of rust fungi. Transcriptome analysis and integrated bioinformatics tools have led to the identification of key genetic determinants of host susceptibility to infection by rusts. Thousands of genes encoding secreted proteins highly expressed during host infection have been reported for different rust species, which represents significant potential towards understanding rust effector function. Recent high-throughput in planta expression screen approaches (effectoromics) have pushed the field ahead even further towards predicting high-priority effectors and identifying avirulence genes. These new insights into rust effector biology promise to inform future research and spur the development of effective and sustainable strategies for managing rust diseases.

Background Conjugation-based episome delivery is a highly efficient method used to transfer DNA into the diatom Phaeodactylum tricornutum , facilitating the production of recombinant proteins and high-value metabolites. However, previous reports have indicated phenotypic heterogeneity among individual cells from clonally propagated exconjugant cell lines, potentially affecting the stability of recombinant protein production in the diatom. Results Here, we characterized the differences between subpopulations with distinct fluorescence intensity phenotypes derived from a single exconjugant colony of P. tricornutum expressing the enhanced green fluorescent protein ( eGFP ). We analyzed the expression cassette sequence integrity, plasmid copy number, and global gene expression. Our findings reveal that lower copy numbers and the deletion of the expression cassette in part of the population contributed to low transgene expression. Gene co-expression analysis identified a set of genes with similar expression pattern to eGFP including a gene encoding a putative Flp recombinase, which may be related to variations in fluorescence intensity. These genes thus present themselves as potential candidates for increasing recombinant proteins production in P. tricornutum episomal expression system. Conclusions Overall, our study elucidates genetic and transcriptomic differences between distinct subpopulations in a clonally propagated culture, contributes to a better understanding of heterogeneity in diatom expression systems for synthetic biology applications.

Chaga (Inonotus obliquus) is a medicinal fungus used in traditional medicine of Native American and North Eurasian cultures. Several studies have demonstrated the medicinal properties of chaga’s bioactive molecules. For example, several terpenoids (e.g., betulin, betulinic acid and inotodiol) isolated from I. obliquus cells have proven effectiveness in treating different types of tumor cells. However, the molecular mechanisms and regulation underlying the biosynthesis of chaga terpenoids remain unknown. In this study, we report on the optimization of growing conditions for cultured I. obliquus in presence of different betulin sources (e.g., betulin or white birch bark). It was found that better results were obtained for a liquid culture pH 6.2 at 28 °C. In addition, a de novo assembly and characterization of I. obliquus transcriptome in these growth conditions using Illumina technology was performed. A total of 219,288,500 clean reads were generated, allowing for the identification of 20,072 transcripts of I. obliquus including transcripts involved in terpenoid biosynthesis. The differential expression of these genes was confirmed by quantitative-PCR. This study provides new insights on the molecular mechanisms and regulation of I. obliquus terpenoid production. It also contributes useful molecular resources for gene prediction or the development of biotechnologies for the alternative production of terpenoids.

Melampsora larici-populina (Mlp) is a devastating pathogen of poplar trees, causing the defoliating poplar leaf rust disease. Genomic studies have revealed that Mlp possesses a repertoire of 1184 small secreted proteins (SSPs), some of them being characterized as candidate effectors. However, how they promote virulence is still unclear. This study investigates the candidate effector Mlp37347’s role during infection. We developed a stable Arabidopsis transgenic line expressing Mlp37347 tagged with the green fluorescent protein (GFP). We found that the effector accumulated exclusively at plasmodesmata (PD). Moreover, the presence of the effector at plasmodesmata favors enhanced plasmodesmatal flux and reduced callose deposition. Transcriptome profiling and a gene ontology (GO) analysis of transgenic Arabidopsis plants expressing the effector revealed that the genes involved in glucan catabolic processes are up-regulated. This effector has previously been shown to interact with glutamate decarboxylase 1 (GAD1), and in silico docking analysis supported the strong binding between Mlp37347 and GAD1 in this study. In infection assays, the effector promoted Hyalonoperospora arabidopsidis growth but not bacterial growth. Our investigation suggests that the effector Mlp37347 targets PD in host cells and promotes parasitic growth.

Rust fungi cause epidemics that threaten the production of important plant species, such as wheat and soy. Melampsora larici-populina (Mlp) causes the poplar rust and encodes at least 1184 candidate effectors (CEs) whose functions are poorly known. In this study, we sequenced the transcriptome and used mass spectrometry to analyze the metabolome of Arabidopsis plants constitutively expressing 14 Mlp CEs and of a control line to discover alterations leading to plant susceptibility. We found 2299 deregulated genes across the experiment. Genes involved in pattern-triggered immunity, such as FRK1, PR1, RBOHD, and WRKY33, as well as AUX/IAA genes were down-regulated. We further observed that 680 metabolites were deregulated in at least one CE-expressing transgenic line, with “highly unsaturated and phenolic compounds” and “peptides” enriched among down- and up-regulated metabolites. Interestingly, transgenic lines expressing unrelated CEs had correlated patterns of gene and metabolite deregulation, while expression of CEs belonging to the same family deregulated different genes and metabolites. Thus, our results uncouple effector sequence similarity and function. This supports that effector functional investigation in the context of their virulence activity and effect on plant susceptibility requires the investigation of the individual effector and precludes generalization based on sequence similarity.

Summary Amaryllidaceae alkaloids (AAs) are diverse bioactive metabolites with significant pharmaceutical potential, derived from 4′‐O‐methylnorbelladine (4′OM). The biosynthesis of these compounds involves the condensation of tyramine and 3,4‐dihydroxybenzaldehyde by norbelladine synthase (NBS) and/or noroxomaritidine/norcraugsodine reductase (NR), followed by O‐methylation. Cytochrome P450 enzymes, particularly the CYP96T family, introduce further structural diversity through C–C couplings, resulting in lycorine, galanthamine and crinine cores. Despite their importance, the exact biosynthetic pathways remain poorly defined. In this study, we describe key enzymes from Leucojum aestivum (La), providing crucial insight into AA biosynthesis. Transient expression in Nicotiana benthamiana demonstrated that LaNBS and LaNRII catalyse the conversion of tyramine and 3,4‐dihydroxybenzaldehyde to norbelladine, which is subsequently O‐methylated by a norbelladine‐4′‐O‐methyltransferase (LaN4′OMT) in planta. Co‐agroinfiltration of LaNBS, LaNRII, LaN4′OMT and LaCYP96T1 resulted in the production of various phenol‐coupled products, with lycorine as the predominant compound, alongside haemanthamine, crinine/vittatine and norgalanthamine. This study identifies LaCYP96T1 and LaCYP96T2 as the first monocot enzymes capable of catalysing all three regioselective C‐C phenol couplings and also highlights the substrate promiscuity of LaNRII. The findings not only elucidate critical steps in AA biosynthesis but also open new avenues for biotechnological application in producing valuable alkaloids, offering potential for novel drug development. Schematic representation of Amaryllidaceae alkaloid biosynthesis in Leucojum aestivum. Norbelladine synthase (LaNBS) and noroxomaritidine/norcraugsodine reductase (LaNRII) catalyze the condensation of tyramine and 3,4‐dihydroxybenzaldehyde to form norbelladine, which is subsequently O‐methylated by norbelladine‐4′‐O‐methyltransferase (LaN4′OMT). Cytochrome P450 enzymes LaCYP96T1 and LaCYP96T2 mediate C–C phenol couplings, leading to the formation of lycorine, haemanthamine, crinine/vittatine, and norgalanthamine. Transient expression in Nicotiana benthamiana confirms enzymatic activities and alkaloid production, providing new insights into Amaryllidaceae alkaloid biosynthesis and biotechnological applications.

The basidiomycete Melampsora larici-populina causes poplar rust disease by invading leaf tissues and secreting effector proteins through specialized infection structures known as haustoria. The mechanisms by which rust effectors promote pathogen virulence are poorly understood. The present study characterized Mlp124478, a candidate effector of M. larici-populina. We used the models Arabidopsis thaliana and Nicotiana benthamiana to investigate the function of Mlp124478 in plant cells. We established that Mlp124478 accumulates in the nucleus and nucleolus, however its nucleolar accumulation is not required to promote growth of the oomycete pathogen Hyaloperonospora arabidopsidis. Stable constitutive expression of Mlp124478 in A. thaliana repressed the expression of genes involved in immune responses, and also altered leaf morphology by increasing the waviness of rosette leaves. Chip-PCR experiments showed that Mlp124478 associats'e with the TGA1a-binding DNA sequence. Our results suggest that Mlp124478 exerts a virulence activity and binds the TGA1a promoter to suppress genes induced in response to pathogen infection.

Background Pharmaceutical safety is an increasing global priority, particularly as the demand for therapeutic compounds rises alongside population growth. Phytocannabinoids, a class of bioactive polyketide molecules derived from plants, have garnered significant attention due to their interaction with the human endocannabinoid system, offering potential benefits for managing a range of symptoms and conditions. Traditional extraction from cannabis plants poses regulatory, environmental, and yield-related challenges. Consequently, microbial biosynthesis has emerged as a promising biotechnological alternative to produce cannabinoids in a controlled, scalable, and sustainable manner. Developing diatom-based biofactories represent a crucial step in advancing this biotechnology, enabling the efficient production of high-valued compounds such as cannabinoids. Results We engineered the diatom Phaeodactylum tricornutum , a unicellular photosynthetic model organism prized for its naturally high lipid content, to produce olivetolic acid (OA), a key metabolic precursor to most cannabinoids. The genes encoding tetraketide synthase and olivetolic acid cyclase from cannabis were cloned onto episomal vectors and introduced using bacterial conjugation in two separate P. tricornutum transconjugant lines to evaluate enzyme activity and OA production in vivo. Both genes were successfully expressed, and the corresponding enzymes accumulated within the transconjugant lines. However, despite testing the cell extracts individually and in combination, OA accumulation was not detected suggesting potential conversion or utilization of OA by endogenous metabolic pathways within the diatoms. To investigate this further, we analyzed the impact of Cs TKS expression on the diatom’s metabolome, revealing significant alterations that may indicate metabolic flux redirection or novel pathway interactions. Conclusions Our study demonstrates the successful expression of cannabinoid biosynthetic genes in P. tricornutum but highlights challenges in OA accumulation, likely due to endogenous metabolic interactions. These findings underscore the complexity of metabolic engineering in diatoms and suggest the need for further pathway optimization and metabolic flux analysis to achieve efficient cannabinoid biosynthesis. This research contributes to advancing sustainable biotechnological approaches for cannabinoid production. Graphical abstract