Agrobacterium effector protein VirE2 is important for plant transformation. VirE2 likely coats transferred DNA (T-DNA) in the plant cell and protects it from degradation. VirE2 localizes to the plant cytoplasm and interacts with several host proteins. Plant-expressed VirE2 can complement a virE2 mutant Agrobacterium strain to support transformation. We investigated whether VirE2 could facilitate transformation from a nuclear location by affixing to it a strong nuclear localization signal (NLS) sequence. Only cytoplasmic-, but not nuclear-localized, VirE2 could stimulate transformation. To investigate the ways VirE2 supports transformation, we generated transgenic Arabidopsis plants containing a virE2 gene under the control of an inducible promoter and performed RNA-seq and proteomic analyses before and after induction. Some differentially expressed plant genes were previously known to facilitate transformation. Knockout mutant lines of some other VirE2 differentially expressed genes showed altered transformation phenotypes. Levels of some proteins known to be important for transformation increased in response to VirE2 induction, but prior to or without induction of their corresponding mRNAs. Overexpression of some other genes whose proteins increased after VirE2 induction resulted in increased transformation susceptibility. We conclude that cytoplasmically localized VirE2 modulates both plant RNA and protein levels to facilitate transformation.
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Signal Peptides Generated by Attention-Based Neural Networks
Short (15–30 residue) chains of amino acids at the amino termini of expressed proteins known as signal peptides (SPs) specify secretion in living cells. We trained an attention-based neural network, the Transformer model, on data from all available organisms in Swiss-Prot to generate SP sequences. Experimental testing demonstrates that the model-generated SPs are functional: when appended to enzymes expressed in an industrial Bacillus subtilis strain, the SPs lead to secreted activity that is competitive with industrially used SPs. Additionally, the model-generated SPs are diverse in sequence, sharing as little as 58% sequence identity to the closest known native signal peptide and 73% ± 9% on average.
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- Award ID(s):
- 1937902
- NSF-PAR ID:
- 10179180
- Date Published:
- Journal Name:
- ACS Synthetic Biology
- ISSN:
- 2161-5063
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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ABSTRACT Plant pathogens utilize a portfolio of secreted effectors to successfully infect and manipulate their hosts. It is, however, still unclear whether changes in secretomes leading to host specialization involve mostly effector gene gains/losses or changes in their sequences. To test these hypotheses, we compared the secretomes of three host-specific castrating anther smut fungi ( Microbotryum ), two being sister species. To address within-species evolution, which might involve coevolution and local adaptation, we compared the secretomes of strains from differentiated populations. We experimentally validated a subset of signal peptides. Secretomes ranged from 321 to 445 predicted secreted proteins (SPs), including a few species-specific proteins (42 to 75), and limited copy number variation, i.e., little gene family expansion or reduction. Between 52% and 68% of the SPs did not match any Pfam domain, a percentage that reached 80% for the small secreted proteins, indicating rapid evolution. In comparison to background genes, we indeed found SPs to be more differentiated among species and strains, more often under positive selection, and highly expressed in planta ; repeat-induced point mutations (RIPs) had no role in effector diversification, as SPs were not closer to transposable elements than background genes and were not more RIP affected. Our study thus identified both conserved core proteins, likely required for the pathogenic life cycle of all Microbotryum species, and proteins that were species specific or evolving under positive selection; these proteins may be involved in host specialization and/or coevolution. Most changes among closely related host-specific pathogens, however, involved rapid changes in sequences rather than gene gains/losses. IMPORTANCE Plant pathogens use molecular weapons to successfully infect their hosts, secreting a large portfolio of various proteins and enzymes. Different plant species are often parasitized by host-specific pathogens; however, it is still unclear whether the molecular basis of such host specialization involves species-specific weapons or different variants of the same weapons. We therefore compared the genes encoding secreted proteins in three plant-castrating pathogens parasitizing different host plants, producing their spores in plant anthers by replacing pollen. We validated our predictions for secretion signals for some genes and checked that our predicted secreted proteins were often highly expressed during plant infection. While we found few species-specific secreted proteins, numerous genes encoding secreted proteins showed signs of rapid evolution and of natural selection. Our study thus found that most changes among closely related host-specific pathogens involved rapid adaptive changes in shared molecular weapons rather than innovations for new weapons.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1021/acssynbio.0c00219
