Fusarium verticillioides induces maize-derived ethylene to promote virulence by engaging fungal G-protein signaling.
Molecular plant-microbe interactions : MPMI
Seed maceration and contamination with mycotoxin fumonisin inflicted by Fusarium verticillioides is major disease of concern for maize producers world-wide. Meta-analyses of QTL for Fusarium ear rot resistance uncovered several ethylene (ET) biosynthesis and signaling genes within them, implicating ET in maize interactions with F. verticillioides. We tested this hypothesis using maize knock-out mutants of the 1-aminocyclopropane-1-carboxylate (ACC) synthases, ZmACS2 and ZmACS6. Infected wild-type seed emitted five-fold higher ET levels compared to controls, whereas ET was abolished in the acs2 and acs6 single and double mutants. The mutants supported reduced fungal biomass, conidia and fumonisin content. Normal susceptibility was restored in the acs6 mutant with exogenous treatment of ET precursor, ACC. Subsequently, we showed that fungal G-protein signaling is required for virulence via induction of maize-produced ET. F. verticillioides Gbeta subunit and two regulators of G-protein signaling mutants displayed reduced seed colonization and decreased ET levels. These defects were rescued by exogenous application of ACC. We concluded that pathogen-induced ET facilitates F. verticillioides colonization of seed, and in turn host ET production is manipulated via G-protein signaling of F. verticillioides to facilitate pathogenesis.
View details for DOI 10.1094/MPMI-09-20-0250-R
View details for PubMedID 34165327
RNA silencing suppressor-influenced performance of a virus vector delivering both guide RNA and Cas9 for CRISPR gene editing.
2021; 11 (1): 6769
We report on further development of the agroinfiltratable Tobacco mosaic virus (TMV)-based overexpression (TRBO) vector to deliver CRISPR/Cas9 components into plants. First, production of a Cas9 (HcoCas9) protein from a binary plasmid increased when co-expressed in presence of suppressors of gene silencing, such as the TMV 126-kDa replicase or the Tomato bushy stunt virus P19 protein. Such suppressor-generated elevated levels of Cas9 expression translated to efficient gene editing mediated by TRBO-G-3'gGFP expressing GFP and also a single guide RNA targeting the mgfp5 gene in the Nicotiana benthamiana GFP-expressing line 16c. Furthermore, HcoCas9 encoding RNA, a large cargo insert of 4.2kb, was expressed from TRBO-HcoCas9 to yield Cas9 protein again at higher levels upon co-expression with P19. Likewise, co-delivery of TRBO-HcoCas9 and TRBO-G-3'gGFP in the presence of P19 also resulted in elevated levels percentages of indels (insertions and deletions). These data also revealed an age-related phenomenon in plants whereby the RNA suppressor P19 had more of an effect in older plants. Lastly, we used a single TRBO vector to express both Cas9 and a sgRNA. Taken together, we suggest that viral RNA suppressors could be used for further optimization of single viral vector delivery of CRISPR gene editing parts.
View details for DOI 10.1038/s41598-021-85366-4
View details for PubMedID 33762584
Native processing of sgRNA transcripts to create catalytic Cas9/sgRNA complexes in planta.
The current CRISPR/Cas9 gene editing dogma for single guide RNA (sgRNA) delivery is based on the premise that 5'- and 3'-nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides at the 5' and 3' termini of sgRNAs. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana. This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5', but not 3', proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5' overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript, it also produced indels when delivered with Cas9. These results reveal that 5' "auto-processing" of progenitor sgRNAs occurs natively in plants. Towards a possible mechanism for the perceived "auto-processing", we found, using in vitro-generated RNAs and those isolated from plants, that the 5' to 3' exoribonuclease XRN1 can degrade elongated progenitor sgRNAs whereas the mature sgRNA end products are resistant. Comparisons with other studies suggest that sgRNA "auto-processing" may be a phenomenon not unique to plants, but present in other eukaryotes as well.
View details for DOI 10.1104/pp.20.00150
View details for PubMedID 32665336
Plant Virus Vectors 3.0: Transitioning into Synthetic Genomics.
Annual review of phytopathology
Plant viruses were first implemented as heterologous gene expression vectors more than three decades ago. Since then, the methodology for their use has varied, but we propose it was the merging of technologies with virology tools, which occurred in three defined steps discussed here, that has driven viral vector applications to date. The first being the advent of molecular biology and reverse genetics, which enabled the cloning and manipulation of viral genomes to express genes of interest (vectors 1.0). The second stems from the discovery of RNA silencing and the development of high-throughput sequencing technologies that allowed the convenient and widespread use of virus-induced gene silencing (vectors 2.0). Here, we briefly review the events that led to these applications, but this treatise mainly concentrates on the emerging versatility of gene-editing tools, which has enabled the emergence of virus-delivered genetic queries for functional genomics and virology (vectors 3.0).
View details for DOI 10.1146/annurev-phyto-082718-100301
View details for PubMedID 31185187