Richard W. Michelmore

Aflatoxins produced by some species of Aspergillus pose a significant threat to global food safety, because they contaminate a variety of crops including maize. In this study, we used host-induced gene silencing (HIGS) to reduce aflatoxin accumulation in maize upon infection with Aspergillus flavus and Aspergillus parasiticus . Two RNAi constructs were designed and introduced into maize, one targeting two essential fungal genes involved in ergosterol biosynthesis ( CYP51A and CYP51B ) and the other targeting three aflatoxin biosynthesis genes ( AflC , AflD , and AflR ). The presence of RNAi transgenes was confirmed in 17 and 14 independent lines possessing the CYP51A/CYP51B RNAi construct and the AflC/AflD/AflR RNAi construct, respectively. Most transgenic lines had single-locus integrations and one or two copies of the transgene. Transgene expression varied considerably among the lines. Small RNA sequencing confirmed the production of siRNAs from the HIGS trigger sequences. Levels of CYP51A , AflC , AflD, and AflR transcripts in Aspergillus spp. were successfully decreased upon infection of transgenic maize. However, significant reduction in the level of CYP51B transcripts was not achieved and the CYP51A/CYP51B RNAi transgene had no discernible effect on fungal growth in infected maize. In contrast, we observed significant reductions in aflatoxin accumulation in four AflC/AflD/AflR RNAi transgenic lines infected with A. flavus and in two transgenic lines infected with A. parasiticus . Notably, one AflC/AflD/AflR transgenic line exhibited minimal aflatoxin contamination upon kernel infection with A. parasiticus . These results demonstrate the potential of HIGS to reduce aflatoxin production in mature corn kernels.

Background: Strigolactones (SLs) regulate diverse aspects of plant development and have been implicated in promoting leaf senescence. However, senescence phenotypes associated with SL deficiency have not been consistently observed across species, suggesting that this function may be species- or context-dependent. Moreover, the contribution of endogenous SL biosynthesis to senescence in leafy vegetable crops remains unclear. Here, we investigated the role of the SL biosynthetic gene MORE AXILLARY GROWTH1 (MAX1) in dark-induced leaf senescence in lettuce ( Lactuca sativa ). Results: We found that endogenous SL biosynthesis plays a major role in dark-induced senescence in lettuce. SL pathway genes were induced during dark storage, while exogenous GR24 accelerated senescence and lettuce MAX1 (LsMAX1) complemented the delayed-senescence phenotype of the Arabidopsis max1 mutant. Consistent with these findings, CRISPR/Cas9-generated Lsmax1 mutants exhibited a pronounced stay-green phenotype during prolonged darkness, accompanied by strongly reduced induction of key senescence-associated genes. Despite this delayed visible senescence, Lsmax1 retained a substantial transcriptional response to dark storage. Strikingly, loss of LsMAX1 did not simply weaken the wild-type senescence program, but redirected part of the response toward a distinct stress-associated transcriptional state that was largely absent from wild type. Loss of LsMAX1 did not affect vegetative rosette architecture, although increased branching emerged after bolting. Conclusions: Our findings establish MAX1-dependent SL biosynthesis as an important regulator of leaf senescence in lettuce and reveal a role that extends beyond controlling the rate of senescence. Rather than simply delaying the wild-type program, loss of LsMAX1 alters the transcriptional trajectory of senescence, favoring an alternative stress-associated state during prolonged darkness. The strong stay-green phenotype without detectable changes to vegetative rosette architecture further highlights SL biosynthesis as a potential target for extending postharvest longevity in lettuce and other leafy crops.

Variegation plays important biological roles and provides sensory quality for ornamental crops. The mechanisms of variegation have been well studied. However, the molecular mechanism underlying the density of variegation remains largely unknown. In this study, we genetically mapped and cloned a gene negatively controlling the density of red spots on lettuce leaves. The causal gene, Sparse Red Spots (SRS), encodes a C2H2 zinc finger protein. SRS interacted physically with a plastid ribosomal protein L15 (LsRPL15). Knocking down LsRPL15 converted dense spots to sparse spots. SRS controlled the spot density by indirectly regulating the stochastic expression of RLL2AV, a MYB-encoding gene. A SUVH-INTERACTING DNAJ DOMAIN-CONTAINING PROTEIN (LsSDJ) directly bound to the promoter of RLL2AV. Knockout of LsSDJ in densely spotted lettuce dramatically decreased spot density. Yeast two-hybrid and dual-luciferase reporter assays revealed that LsSDJ interacted with LsTCP2 to activate RLL2AV. Further analysis demonstrated that LsRPL15 interacted with the LsSDJ-LsTCP2 module in vivo. SRS interacted with LsRPL15 physically to suppress the stochastic activation rate of RLL2AV, generating sparse spots. Conversely, a natural point mutation leading to an amino acid substitution in SRS abolished its interaction with LsRPL15, thereby releasing the positive regulatory effect of LsRPL15 on spot density, which enhanced the activation rate of RLL2AV, leading to dense spots. This study elucidated a mechanism underlying the spot density and provides new insights into the regulation of gene expression pattern.

Plants rely on cell surface immune receptors to detect microbial patterns and initiate effective defense responses. Although the Asteraceae family is one of the largest and economically important plant groups, little information is available about its pattern-triggered immunity signaling. Cultivated lettuce ( Lactuca sativa L.) recognizes a 24-amino acid peptide (nlp24) from necrosis- and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) found in bacteria, fungi, and oomycetes. Here, we perform an extensive characterization of nlp24-induced immune responses in lettuce and identify the LETTUCE nlp24 RECEPTOR (LNR) as the leucine-rich repeat receptor-like kinase mediating its recognition. Remarkably, nlp24 recognition in lettuce and subsequent activation of defenses strongly depend on the conserved heptapeptide motif (GHRHDWE). Structural modeling-guided mutagenesis experiments suggest that residues in the nlp24 heptapeptide interact with a hydrophobic pocket in the LNR solenoid structure. Divergent ligand specificities and the absence of sequence homology between LNR and the Arabidopsis nlp24-recognizing receptor RLP23 indicate that NLP recognition in lettuce and Arabidopsis emerged independently, through convergent evolution. Our phylogenetic analysis shows that LNR is closely related to Arabidopsis MALE DISCOVERER 1-INTERACTING RECEPTOR-LIKE KINASE 2 (MIK2) but belongs to a distinct, Asteraceae-specific monophyletic subgroup that has undergone a significant expansion in Lactuca . Our findings provide insights into the mechanisms of pattern-triggered immunity in lettuce and present an example of convergent evolution of plant pattern-recognition receptors.

The Asteraceae (Compositae) is the largest flowering plant family, ubiquitous in most terrestrial communities, and morphologically diverse. A two-step, ancient whole genome triplication (paleohexaploidization) occurred at approximately the same time as the evolutionary innovation and adaptive radiation of the family during the middle Eocene. Despite its importance, the consequences of this triplication have yet to be tracked in context of the Asteraceae genome evolution. To do so, we applied a synteny oriented phylogenomic analysis of 23 Asterales genomes. We identified 16 genomic groups that date back to the common diploid ancestor of all Asteraceae. Each group underwent triplication, resulting in 48 genomic blocks (16 × 3) that collectively represent the ancestral Asteraceae genome, excluding the early-diverging lineages which do not share the second step. We then analyzed the evolutionary dynamics of the 48 genomic blocks across the Asteraceae phylogeny. We found that modern Asteraceae genomes are genetic mosaics of three progenitor genomes, shaped by genomic exchanges, chromosomal rearrangements, and gene fractionation. One hundred fifty-seven genes retained three paleohexaploid-derived syntenic paralogs across most Asteraceae species. Transcription factors and auxin-related genes are significantly overrepresented in these triplets, and expression of the paleohexaploidy paralogs is spatiotemporally differentiated. These genes are involved in the development of floral capitula, a remarkable morphological innovation of the family. The discovery of the 157 triplicated genes can direct further study to understand the evolutionary innovation, and the synteny-phylogenomic framework provides a comparative framework to characterize newly sequenced Asteraceae genomes.

Ascorbic acid (AsA) is a key antioxidant and nutrient in plants, regulating reactive oxygen species (ROS) levels and maintaining cellular redox homeostasis. The AsA recycling pathway sustains AsA pools by restoring its oxidized forms, ensuring intracellular balance. Among the enzymes involved, monodehydroascorbate reductase (MDHAR) is important for the regeneration of AsA from monodehydroascorbate. In this study, we analyzed the four MDHAR paralogs in Lactuca sativa using CRISPR/Cas9 to determine whether disruption of individual MDHAR genes could alter AsA levels in lettuce leaves. Unexpectedly, none of the knockouts caused long-term changes in leaf AsA content. Transcriptomic analyses at 14 and 28 days showed minimal effects on AsA recycling or biosynthesis genes, except MDHAR genes. However, several other genes indirectly implicated in AsA regulation displayed differential expression in all mutants compared to the wild type, suggesting the presence of a complex regulatory network. In particular, genes encoding transcription factors (TFs), such as mTERF15, COL9, UPBEAT1, NAC28, and NAC42, were differentially regulated in all MDHAR mutants compared to the wild type at 28 days. These findings indicate that, although AsA content remains unchanged, MDHAR single knockouts alter expression of other genes through which the plants may indirectly compensate to maintain redox homeostasis.

With the current speed of sequencing, there is a desire for standardized and automated genome assembly and annotation to produce high-quality genomes as input for comparative (pan)genomics. Therefore, we created a convenience pipeline using existing tools that creates annotated genome assemblies from HiFi (and optionally ultra-long ONT and/or Hi-C) reads for a set of related individuals as well as a related reference genome. Our pipeline is species-agnostic and generates an extensive quality assessment report that can be used for manual filtering and refinement of the assembly and annotation. It includes statistics for individual completeness and contamination assessments as well as a concise pangenome view. The pipeline is implemented in Snakemake and available with a GPLv3 licence at GitHub under github.com/dirkjanvw/MoGAAAP, at Zenodo under doi.org/10.5281/zenodo.14833021, and can be installed through Bioconda.

The success of many engineered crop traits depends on the stable expression of transgenes, but their effectiveness is frequently at risk due to transgene silencing. The reason why certain transgenes are targeted by silencing pathways while others remain highly expressed and durable has remained a major question for decades due to the lack of technologies to study the initiation of transgene silencing. We developed 2 technologies to identify the trigger of transgene silencing in Arabidopsis (Arabidopsis thaliana) and in lettuce (Latuca sativa): one using the RUBY transgene to visualize the precise developmental time point of transgene silencing and the second to identify all transcripts produced from a transgene. By combining these 2 methods with Machine Learning, we identified an aberrant transgene-derived RNA that accumulates to high levels and closely correlates with the onset of transgene silencing. Our data suggest that a ribosome stalled at an unusual 3-consecutive-histidine peptide sequence on the RUBY transcript triggers No-Go RNA Decay and cleavage of the RUBY mRNA. The production of this cleaved aberrant RNA precedes RNA interference during the triggering of transgene silencing; it is innate to the transgene coding sequence, independent of the promoter used or whether it is transformed into a model plant or crop.

With the current speed of sequencing, there is a desire for standardised and automated genome assembly and annotation to produce high-quality genomes as input for comparative (pan)genomics. Therefore, we created a convenience pipeline using existing tools that creates annotated genome assemblies from HiFi (and optionally ultra-long ONT and/or Hi-C) reads for a set of related accessions as well as a related reference genome. Our pipeline is species-agnostic and generates an extensive quality assessment report that can be used for manual filtering and refinement of the assembly and annotation. It includes statistics for individual completeness and contamination assessments as well as a concise pangenome view. The pipeline is implemented in Snakemake and available with a GPLv3 license at GitHub under github.com/dirkjanvw/MoGAAAP and at Zenodo under doi.org/10.5281/zenodo.14833021.