Genomic consequences associated with Agrobacterium-mediated transformation of plants.

Attenuated strains of the naturally occurring plant pathogen Agrobacterium tumefaciens can transfer virtually any DNA sequence of interest to model plants and crops. This has made Agrobacterium-mediated transformation (AMT) one of the most commonly used tools in agricultural biotechnology. Understanding AMT, and its functional consequences, is of fundamental importance given that it sits at the intersection of many fundamental fields of study, including plant-microbe interactions, DNA repair/genome stability, and epigenetic regulation of gene expression. Despite extensive research and use of AMT over the last 40 years, the extent of genomic disruption associated with integrating exogenous DNA into plant genomes using this method remains underappreciated. However, new technologies like long-read sequencing make this disruption more apparent, complementing previous findings from multiple research groups that have tackled this question in the past. In this review, we cover progress on the molecular mechanisms involved in Agrobacterium-mediated DNA integration into plant genomes. We also discuss localized mutations at the site of insertion and describe the structure of these DNA insertions, which can range from single copy insertions to large concatemers, consisting of complex DNA originating from different sources. Finally, we discuss the prevalence of large-scale genomic rearrangements associated with the integration of DNA during AMT with examples. Understanding the intended and unintended effects of AMT on genome stability is critical to all plant researchers who use this methodology to generate new genetic variants.

Deletions within intronic T-DNA lead to reversion of T-DNA mutant phenotypes.

Agrobacterium-mediated transformation enables random transfer-DNA (T-DNA) insertion into plant genomes. T-DNA insertion into a gene's exons, introns or untranscribed regions close to the start or stop codon can disrupt gene function. Such T-DNA mutants have been useful for reverse genetics analysis, especially in Arabidopsis thaliana. As T-DNAs are inserted into genomic DNA, they are generally believed to be stably inherited. Here, we report a phenomenon of reversion of intronic T-DNA mutant phenotypes. From a suppressor screen using intronic T-DNA pi4kß1,2 double mutant, we recovered intragenic mutants of pi4kß1, which suppressed the autoimmunity of the double mutant. These mutants carried deletions in the intronic T-DNAs, resulting in elevated transcription of normal PI4Kß1. Such reversion of T-DNA insertional mutant phenotype stresses the need for caution when using intronic T-DNA mutants and reiterates the importance of using irreversible null mutant alleles in genetic analyses.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A. thaliana deficient in polymerase theta (Polθ), a core component of theta-mediated end joining (TMEJ) of DNA breaks, have led to the suggestion of a direct involvement of this enzyme in the homology-directed process. Here, by monitoring homology-driven gene conversion in plants with CRISPR reagent and donor sequences pre-integrated at random sites in the genome (in planta GT), we demonstrate that Polθ action is not required for GT, but instead suppresses the process, likely by promoting the repair of the DNA break by end-joining. This finding indicates that lack of donor integration explains the previously established reduced GT rates seen upon transformation of Polθ-deficient plants. Our study additionally provides insight into ectopic gene targeting (EGT), recombination events between donor and target that do not map to the target locus. EGT, which occurs at similar frequencies as "true" GT during transformation, was rare in our in planta GT experiments arguing that EGT predominantly results from target locus recombination with nonintegrated T-DNA molecules. By describing mechanistic features of GT our study provides directions for the improvement of precise genetic modification of plants.

Screening of pathogenicity-deficient Penicillium italicum mutants established by Agrobacterium tumefaciens-mediated transformation.

Blue mold, caused by Penicillium italicum, is one of the main postharvest diseases of citrus fruits during storage and marketing. The pathogenic mechanism remains largely unclear. To explore the potential pathogenesis-related genes of this pathogen, a T-DNA insertion library of P. italicum PI5 was established via Agrobacterium tumefaciens-mediated transformation (ATMT). The system yielded 200-250 transformants per million conidia, and the transformants were genetically stable after five generations of successive subcultures on hygromycin-free media. 2700 transformants were obtained to generate a T-DNA insertion library of P. italicum. Only a few of the 200 randomly selected mutants exhibited significantly weakened virulence on citrus fruits, with two mutants displaying attenuated sporulation. The T-DNA in the two mutants existed as a single copy. Moreover, the mutant genes PiBla (PITC_048370) and PiFTF1 (PITC_077280) identified may be involved in conidia production by regulating expressions of the key regulatory components for conidiogenesis. These results demonstrated that the ATMT system is useful to obtain mutants of P. italicum for further investigation of the molecular mechanisms of pathogenicity and the obtained two pathogenesis-related genes might be novel loci associated with pathogenesis and conidia production.

A word of caution: T-DNA-associated mutagenesis in plant reproduction research.

T-DNA transformation is prevalent in Arabidopsis research and has expanded to a broad range of crops and model plants. While major progress has been made in optimizing the Agrobacterium-mediated transformation process for various species, a variety of pitfalls associated with the T-DNA insertion may lead to the misinterpretation of T-DNA mutant analysis. Indeed, secondary mutagenesis either on the integration site or elsewhere in the genome, together with epigenetic interactions between T-DNA inserts or frequent genomic rearrangements, can be tricky to differentiate from the effect of the knockout of the gene of interest. These are mainly the case for genomic rearrangements that become balanced in filial generations without consequential phenotypical defects, which may be confusing particularly for studies that aim to investigate fertility and gametogenesis. As a cautionary note to the plant research community studying gametogenesis, we here report an overview of the consequences of T-DNA-induced secondary mutagenesis with emphasis on the genomic imbalance on gametogenesis. Additionally, we present a simple guideline to evaluate the T-DNA-mutagenized transgenic lines to decrease the risk of faulty analysis with minimal experimental effort.

Gene targeting in polymerase theta-deficient Arabidopsis thaliana.

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events. An additional complexity is that the product of recombination between T-DNA and target locus may not only map to the target locus (true GT), but also to random positions in the genome (ectopic GT). In this study, we have investigated how TMEJ functionality affects the biology of GT in plants, by using Arabidopsis thaliana mutated for the TEBICHI gene, which encodes for Polθ. Whereas in TMEJ-proficient plants we predominantly found GT events accompanied by random T-DNA integrations, GT events obtained in the teb mutant background lacked additional T-DNA copies, corroborating the essential role of Polθ in T-DNA integration. Polθ deficiency also prevented ectopic GT events, suggesting that the sequence of events leading up to this outcome requires TMEJ. Our findings provide insights that can be used for the development of strategies to obtain high-quality GT events in crop plants.

Multiple heterochromatin diversification events in the genome of fungus-farming ants: insights from repetitive sequences.

A substantial portion of the eukaryotic genome includes repetitive DNA, which is important for its stability, regulation, and architecture. Fungus-farming ant genomes show remarkable structural rearrangement rates that were necessary for the establishment of their agriculture-based lifestyle, highlighting the relevance of this peculiar group in understanding the repetitive portion of ant genome. Chromosomal banding studies are in accordance with genomic data because they show that repetitive heterochromatic sequences of basal and derivative Attina species are GC-rich, an uncommon trait in Formicidae. To understand the evolutionary dynamics of heterochromatin in Attina, we compared GC-rich heterochromatin patterns between the Paleoattina and Neoattina clades of this subtribe. To this end, we hybridized the Mrel-C0t probe (highly and moderately repetitive DNA) obtained from Mycetomoellerius relictus, Neoattina with GC-rich heterochromatin, in karyotypes of Paleoattina and Neoattina species. Additionally, we mapped the repetitive sequences (GA)15 and (TTAGG)6 in species of the two clades to investigate their organization and evolutionary patterns in the genome of Attina. The Mrel-C0t probe marked the heterochromatin in M. relictus, in other Mycetomoellerius spp., and in species of Mycetarotes, Cyphomyrmex, and Sericomyrmex (Neoattina). In Mycetomoellerius urichii, only pericentromeric heterochromatin was marked with Mrel-C0t. No marking was observed in Paleoattina species or in Atta and Acromyrmex (Neoattina). These results indicated that different evolutionary events led to heterochromatin differentiation in Attina. The most likely hypothesis is that GC-rich heterochromatin arose in the common ancestor of the two clades and accumulated various changes throughout evolution. The sequences (GA)15 and (TTAGG)6 located in euchromatin and telomeres, respectively, showed more homogeneous results among the species.

T-DNA insertion mutagenesis in Penicillium brocae results in identification of an enolase gene mutant impaired in secretion of organic acids and phosphate solubilization.

Penicillium brocae strain P6 is a phosphate-solubilizing fungus isolated from farmland in Guangdong Province, China. To gain better insights into the phosphate solubilization mechanisms of strain P6, a T-DNA insertion population containing approximately 4500 transformants was generated by Agrobacterium tumefaciens-mediated transformation. The transformation procedure was optimized by using a Hybond N membrane for co-cultivation of A. tumefaciens and P. brocae. A mutant impaired in phosphate solubilization (named MT27) was obtained from the T-DNA insertion population. Thermal asymmetric interlaced PCR was then used to identify the nucleotide sequences flanking the T-DNA insertion site. The T-DNA in MT27 was inserted into the fourth exon of an enolase gene, which shows 90.8 % nucleotide identity with enolase mRNA from Aspergillus neoniger. Amino acid sequence homology analysis indicated that the enolase is well conserved among filamentous fungi and Saccharomyces cerevisiae. Complementation tests with the MT27 mutant confirmed that the enolase gene is involved in phosphate solubilization. Analysis of organic acids in culture supernatants indicated reduced levels of oxalic acid and lactic acid for the MT27 mutant compared to the parent strain P6 or the complementation strain. In conclusion, we suggest that the identified enolase gene of P. brocae is involved in production of specific organic acids, which, when secreted, act as phosphate solubilizing agents.

Extensive natural Agrobacterium-induced transformation in the genus Camellia.

MAIN CONCLUSION: The genus Camellia underwent extensive natural transformation by Agrobacterium. Over a period of 15 million years, at least 12 different inserts accumulated in 72 investigated Camellia species. Like a wide variety of other wild and cultivated plants, Camellia species carry cellular T-DNA sequences (cT-DNAs) in their nuclear genomes, resulting from natural Agrobacterium-mediated transformation. Short and long DNA sequencing reads of 435 accessions belonging to 72 Camellia species (representing 12 out of 14 sections) were investigated for the occurrence of cT-DNA insertions. In all, 12 different cT-DNAs were recovered, either completely or partially, called CaTA to CaTL. Divergence analysis of internal cT-DNA repeats revealed that the insertion events span a period from 0.075 to 15 Mio years ago, and yielded an average transformation frequency of one event per 1.25 Mio years. The two oldest inserts, CaTA and CaTD, have been modified by spontaneous deletions and inversions, and by insertion of various plant sequences. In those cases where enough accessions were available (C. japonica, C. oleifera, C. chekiangoleosa, C. sasanqua and C. pitardii), the younger cT-DNA inserts showed a patchy distribution among different accessions of each species, indicating that they are not genetically fixed. It could be shown that Camellia breeding has led to intersectional transfer of cT-DNAs. Altogether, the cT-DNAs cover 374 kb, and carry 47 open reading frames (ORFs). Two Camellia cT-DNA genes, CaTH-orf358 and CaTK-orf8, represent new types of T-DNA genes. With its large number of cT-DNA sequences, the genus Camellia constitutes an interesting model for the study of natural Agrobacterium transformants.

CRISPR/Cas9 and Agrobacterium tumefaciens virulence proteins synergistically increase efficiency of precise genome editing via homology directed repair in plants.

CRISPR/Cas9 genome editing and Agrobacterium tumefaciens-mediated genetic transformation are widely-used plant biotechnology tools derived from bacterial immunity-related systems, each involving DNA modification. The Cas9 endonuclease introduces DNA double-strand breaks (DSBs), and the A. tumefaciens T-DNA is released by the VirD2 endonuclease assisted by VirDl and attached by VirE2, transferred to the plant nucleus and integrated into the genome. Here, we explored the potential for synergy between the two systems and found that Cas9 and three virulence (Vir) proteins achieve precise genome editing via the homology directed repair (HDR) pathway in tobacco and rice plants. Compared with Cas9T (Cas9, VirD1, VirE2) and CvD (Cas9-VirD2) systems, the HDR frequencies of a foreign GFPm gene in the CvDT system (Cas9-VirD2, VirD1, VirE2) increased 52-fold and 22-fold, respectively. Further optimization of the CvDT process with a donor linker (CvDTL) achieved a remarkable increase in the efficiency of HDR-mediated genome editing. Additionally, the HDR efficiency of the three rice endogenous genes ACETOLACTATE SYNTHASE (ALS), PHYTOENE DESATURASE (PDS), and NITROGEN TRANSPORTER 1.1 B (NRT1.1B) increased 24-, 32- and 16-fold, respectively, in the CvDTL system, compared with corresponding Cas9TL (Cas9T process with a donor linker). Our results suggest that collaboration between CRISPR/Cas9 and Agrobacterium-mediated genetic transformation can make great progress towards highly efficient and precise genome editing via the HDR pathway.

ß1,3-galactosyltransferase on chromosome 6 is essential for the formation of Lewis a structure on N-glycan in Oryza sativa.

ß1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of ß1,3-galactose and α1,4-fucose by individual ß1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing ß1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.

Development of an assay system for the analysis of host RISC activity in the presence of a potyvirus RNA silencing suppressor, HC-Pro.

BACKGROUND: To investigate the mechanism of RNA silencing suppression, the genetic transformation of viral suppressors of RNA silencing (VSRs) in Arabidopsis integrates ectopic VSR expression at steady state, which overcomes the VSR variations caused by different virus infections or limitations of host range. Moreover, identifying the insertion of the transgenic VSR gene is necessary to establish a model transgenic plant for the functional study of VSR. METHODS: Developing an endogenous AGO1-based in vitro RNA-inducing silencing complex (RISC) assay prompts further investigation into VSR-mediated suppression. Three P1/HC-Pro plants from turnip mosaic virus (TuMV) (P1/HC-ProTu), zucchini yellow mosaic virus (ZYMV) (P1/HC-ProZy), and tobacco etch virus (TEV) (P1/HC-ProTe) were identified by T-DNA Finder and used as materials for investigations of the RISC cleavage efficiency. RESULTS: Our results indicated that the P1/HC-ProTu plant has slightly lower RISC activity than P1/HC-ProZy plants. In addition, the phenomena are consistent with those observed in TuMV-infected Arabidopsis plants, which implies that HC-ProTu could directly interfere with RISC activity. CONCLUSIONS: In this study, we demonstrated the application of various plant materials in an in vitro RISC assay of VSR-mediated RNA silencing suppression.

Biodiversity of rolB/C-like Natural Transgene in the Genus Vaccinium L. and Its Application for Phylogenetic Studies.

A variety of plant species found in nature contain agrobacterial T-DNAs in their genomes which they transmit in a series of sexual generations. Such T-DNAs are called cellular T-DNAs (cT-DNAs). cT-DNAs have been discovered in dozens of plant genera, and are suggested to be used in phylogenetic studies, since they are well-defined and unrelated to other plant sequences. Their integration into a particular chromosomal site indicates a founder event and a clear start of a new clade. cT-DNA inserts do not disseminate in the genome after insertion. They can be large and old enough to generate a range of variants, thereby allowing the construction of detailed trees. Unusual cT-DNAs (containing the rolB/C-like gene) were found in our previous study in the genome data of two Vaccinium L. species. Here, we present a deeper study of these sequences in Vaccinium L. Molecular-genetic and bioinformatics methods were applied for sequencing, assembly, and analysis of the rolB/C-like gene. The rolB/C-like gene was discovered in 26 new Vaccinium species and Agapetes serpens (Wight) Sleumer. Most samples were found to contain full-size genes. It allowed us to develop approaches for the phasing of cT-DNA alleles and reconstruct a Vaccinium phylogenetic relationship. Intra- and interspecific polymorphism found in cT-DNA makes it possible to use it for phylogenetic and phylogeographic studies of the Vaccinium genus.

Novel Seed Size: A Novel Seed-Developing Gene in Glycine max.

Soybean-seed development is controlled in multiple ways, as in many known regulating genes. Here, we identify a novel gene, Novel Seed Size (NSS), involved in seed development, by analyzing a T-DNA mutant (S006). The S006 mutant is a random mutant of the GmFTL4pro:GUS transgenic line, with phenotypes with small and brown seed coats. An analysis of the metabolomics and transcriptome combined with RT-qPCR in the S006 seeds revealed that the brown coat may result from the increased expression of chalcone synthase 7/8 genes, while the down-regulated expression of NSS leads to small seed size. The seed phenotypes and a microscopic observation of the seed-coat integument cells in a CRISPR/Cas9-edited mutant nss1 confirmed that the NSS gene conferred small phenotypes of the S006 seeds. As mentioned in an annotation on the Phytozome website, NSS encodes a potential DNA helicase RuvA subunit, and no such genes were previously reported to be involved in seed development. Therefore, we identify a novel gene in a new pathway controlling seed development in soybeans.

Analysis of the Candidate Genes and Underlying Molecular Mechanism of P198, an RNAi-Related Dwarf and Sterile Line.

The genome-wide long hairpin RNA interference (lhRNAi) library is an important resource for plant gene function research. Molecularly characterizing lhRNAi mutant lines is crucial for identifying candidate genes associated with corresponding phenotypes. In this study, a dwarf and sterile line named P198 was screened from the Brassica napus (B. napus) RNAi library. Three different methods confirmed that eight copies of T-DNA are present in the P198 genome. However, only four insertion positions were identified in three chromosomes using fusion primer and nested integrated polymerase chain reaction. Therefore, the T-DNA insertion sites and copy number were further investigated using Oxford Nanopore Technologies (ONT) sequencing, and it was found that at least seven copies of T-DNA were inserted into three insertion sites. Based on the obtained T-DNA insertion sites and hairpin RNA (hpRNA) cassette sequences, three candidate genes related to the P198 phenotype were identified. Furthermore, the potential differentially expressed genes and pathways involved in the dwarfism and sterility phenotype of P198 were investigated by RNA-seq. These results demonstrate the advantage of applying ONT sequencing to investigate the molecular characteristics of transgenic lines and expand our understanding of the complex molecular mechanism of dwarfism and male sterility in B. napus.

High-throughput detection of T-DNA insertion sites for multiple transgenes in complex genomes.

BACKGROUND: Genetic engineering of crop plants has been successful in transferring traits into elite lines beyond what can be achieved with breeding techniques. Introduction of transgenes originating from other species has conferred resistance to biotic and abiotic stresses, increased efficiency, and modified developmental programs. The next challenge is now to combine multiple transgenes into elite varieties via gene stacking to combine traits. Generating stable homozygous lines with multiple transgenes requires selection of segregating generations which is time consuming and labor intensive, especially if the crop is polyploid. Insertion site effects and transgene copy number are important metrics for commercialization and trait efficiency. RESULTS: We have developed a simple method to identify the sites of transgene insertions using T-DNA-specific primers and high-throughput sequencing that enables identification of multiple insertion sites in the T1 generation of any crop transformed via Agrobacterium. We present an example using the allohexaploid oil-seed plant Camelina sativa to determine insertion site location of two transgenes. CONCLUSION: This new methodology enables the early selection of desirable transgene location and copy number to generate homozygous lines within two generations.

CRISPR RNA-guided integrase enables high-efficiency targeted genome engineering in Agrobacterium tumefaciens.

Agrobacterium tumefaciens, the causal agent of plant crown gall disease, has been widely used to genetically transform many plant species. The inter-kingdom gene transfer capability made Agrobacterium an essential tool and model system to study the mechanism of exporting and integrating a segment of bacterial DNA into the plant genome. However, many biological processes such as Agrobacterium-host recognition and interaction are still elusive. To accelerate the understanding of this important plant pathogen and further improve its capacity in plant genetic engineering, we adopted a CRISPR RNA-guided integrase system for Agrobacterium genome engineering. In this work, we demonstrate that INsertion of Transposable Elements by Guide RNA-Assisted TargEting (INTEGRATE) can efficiently generate DNA insertions to enable targeted gene knockouts. In addition, in conjunction with Cre-loxP recombination system, we achieved precise deletions of large DNA fragments. This work provides new genetic engineering strategies for Agrobacterium species and their gene functional analyses.

A CCaMK/Cyclops response element in the promoter of Lotus japonicus calcium-binding protein 1 (CBP1) mediates transcriptional activation in root symbioses.

Early gene expression in arbuscular mycorrhiza (AM) and the nitrogen-fixing root nodule symbiosis (RNS) is governed by a shared regulatory complex. Yet many symbiosis-induced genes are specifically activated in only one of the two symbioses. The Lotus japonicus T-DNA insertion line T90, carrying a promoterless uidA (GUS) gene in the promoter of Calcium Binding Protein 1 (CBP1) is exceptional as it exhibits GUS activity in both root endosymbioses. To identify the responsible cis- and trans-acting factors, we subjected deletion/modification series of CBP1 promoter : reporter fusions to transactivation and spatio-temporal expression analysis and screened ethyl methanesulphonate (EMS)-mutagenized T90 populations for aberrant GUS expression. We identified one cis-regulatory element required for GUS expression in the epidermis and a second element, necessary and sufficient for transactivation by the calcium and calmodulin-dependent protein kinase (CCaMK) in combination with the transcription factor Cyclops and conferring gene expression during both AM and RNS. Lack of GUS expression in T90 white mutants could be traced to DNA hypermethylation detected in and around this element. We concluded that the CCaMK/Cyclops complex can contribute to at least three distinct gene expression patterns on its direct target promoters NIN (RNS), RAM1 (AM), and CBP1 (AM and RNS), calling for yet-to-be identified specificity-conferring factors.

Impairment of carotenoid biosynthesis through CAR1 gene mutation results in CoQ10, sterols, and phytoene accumulation in Rhodotorula mucilaginosa.

Red yeasts, mainly included in the genera Rhodotorula, Rhodosporidiobolus, and Sporobolomyces, are renowned biocatalysts for the production of a wide range of secondary metabolites of commercial interest, among which lipids, carotenoids, and other isoprenoids. The production of all these compounds is tightly interrelated as they share acetyl-CoA and the mevalonate pathway as common intermediates. Here, T-DNA insertional mutagenesis was applied to the wild type strain C2.5t1 of Rhodotorula mucilaginosa for the isolation of albino mutants with impaired carotenoids biosynthesis. The rationale behind this approach was that a blockage in carotenoid biosynthetic pathway could divert carbon flux toward the production of lipids and/or other molecules deriving from terpenoid precursors. One characterized albino mutant, namely, strain W4, carries a T-DNA insertion in the CAR1 gene coding for phytoene desaturase. When cultured in glycerol-containing medium, W4 strain showed significant decreases in cell density and fatty acids content in respect to the wild type strain. Conversely, it reached significantly higher productions of phytoene, CoQ10, and sterols. These were supported by an increased expression of CAR2 gene that codes for phytoene synthase/lycopene cyclase. Thus, in accordance with the starting hypothesis, the impairment of carotenoids biosynthesis can be explored to pursue the biotechnological exploitation of red yeasts for enhanced production of secondary metabolites with several commercial applications. KEY POINTS: • The production of lipids, carotenoids, and other isoprenoids is tightly interrelated. • CAR1 gene mutation results in the overproduction of phytoene, CoQ10, and sterols. • Albino mutants are promising tools for the production of secondary metabolites.

Blistering1 Modulates Penicillium expansum Virulence Via Vesicle-mediated Protein Secretion.

The blue mold fungus, Penicillium expansum, is a postharvest apple pathogen that contributes to food waste by rotting fruit and by producing harmful mycotoxins (e.g. patulin). To identify genes controlling pathogen virulence, a random T-DNA insertional library was created from wild-type P. expansum strain R19. One transformant, T625, had reduced virulence in apples, blistered mycelial hyphae, and a T-DNA insertion that abolished transcription of the single copy locus in which it was inserted. The gene, Blistering1, encodes a protein with a DnaJ domain, but otherwise has little homology outside the Aspergillaceae, a family of fungi known for producing antibiotics, mycotoxins, and cheese. Because protein secretion is critical for these processes and for host infection, mass spectrometry was used to monitor proteins secreted into liquid media during fungal growth. T625 failed to secrete a set of enzymes that degrade plant cell walls, along with ones that synthesize the three final biosynthetic steps of patulin. Consequently, the culture broth of T625 had significantly reduced capacity to degrade apple tissue and contained 30 times less patulin. Quantitative mass spectrometry of 3,282 mycelial proteins revealed that T625 had altered cellular networks controlling protein processing in the endoplasmic reticulum, protein export, vesicle-mediated transport, and endocytosis. T625 also had reduced proteins controlling mRNA surveillance and RNA processing. Transmission electron microscopy of hyphal cross sections confirmed that T625 formed abnormally enlarged endosomes or vacuoles. These data reveal that Blistering1 affects internal and external protein processing involving vesicle-mediated transport in a family of fungi with medical, commercial, and agricultural importance.