Spodoptera frugiperda mRNA export factor interacts with and mediates the nuclear import of Autographa californica multiple nucleopolyhedrovirus ORF34 (Ac34).

Autographa californica multiple nucleopolyhedrovirus orf34 (ac34) is one of the unique genes of alphabaculoviruses. For successful alphabaculovirus replication, viral proteins must be transported to the nucleus. Our previous study showed that the nuclear localization of Ac34 was required for optimal production of budded virions. To investigate the mechanism of Ac34 nuclear import, mass spectrometric analysis was performed to identify potential proteins that may be involved in the nuclear import of Ac34. The result indicated that Spodoptera frugiperda mRNA export factor (SfMEF) may interact with Ac34 during baculovirus infection. Co-immunoprecipitation assays confirmed that Ac34 could interact with SfMEF in the absence of other baculovirus proteins. The deletion of ac34 did not affect the subcellular localization of SfMEF; however, knocking down Sfmef prevented the nuclear import of Ac34 in virus-infected cells. The mutations of C116 or C119 in a potential CCCH zinc finger motif (C116-X2-C119-X8-C128-X2-H131) of Ac34 led to an exclusive cytoplasmic distribution of Ac34, in consistent with our previous finding of mutations of C128 or H131 in this motif. Co-immunoprecipitation analysis showed that the above mutations in the potential zinc finger motif disrupted the interaction between Ac34 and SfMEF, and the loss of the interaction resulted in decreased BV production. Our findings demonstrated that SfMEF interacts with and mediates the nuclear import of Ac34, which is a new nucleocytoplasmic transport pathway used by alphabaculovirus to achieve successful viral replication within the nucleus of the infected cells.

AcMNPV PKIP is required for hyperexpression of very late genes and involved in the hyperphosphorylation of the viral basic protein P6.9.

A previous study showed that a mutation in Autographa californica multiple nucleopolyhedrovirus pkip (ac24) led to severe defects in progeny budded virion production and very late gene transcription at non-permissive temperature. To dissect the underlying mechanism, our early study showed that PKIP is associated with nucleocapsid of budded virion and involved in nucleocapsid assembly. However, how pkip affects very late gene transcription has not been determined. In the present study, double-stranded RNA was used to silence pkip expression during virus infection, resulting in the significant reduction of occlusion body production and polyhedrin expression. To find out whether PKIP regulates polyhedrin expression by affecting the transcription of other viral genes for very late gene expression, a comparative transcriptome analysis of viral genes was performed by RNA sequencing and the result showed that silencing pkip specifically down-regulated transcription of very late genes, while the transcription patterns of the viral genes associated with very late gene transcription were not affected. Since PKIP was reported to interact with and stimulate the activity of virus-encoded protein kinase PK1 and PK1 was involved in the hyperphosphorylation of viral basic protein P6.9, which was required for the maximal hyperexpression of very late genes, we sought to determine the association between PKIP and P6.9. Further experiments showed that PKIP interacted with P6.9 during virus infection, and the deletion of pkip resulted in decreased hyperphosphorylation of P6.9. Taken together, our results indicated that PKIP is involved in hyperphosphorylation of P6.9, which in return maybe required for hyperexpression of very late genes.

Construction of a ß-galactosidase-gene-based fusion is convenient for screening candidate genes involved in regulation of pyrrolnitrin biosynthesis in Pseudomonas chlororaphis G05.

In our recent work, we found that pyrrolnitrin, and not phenazines, contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in the regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-ß-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in the regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

Pyrrolnitrin is more essential than phenazines for Pseudomonas chlororaphis G05 in its suppression of Fusarium graminearum.

Fusarium graminearum is the major causal agent of Fusarium head blight (FHB) disease in cereal crops worldwide. Infection with this fungal phytopathogen can regularly cause severe yield and quality losses and mycotoxin contamination in grains. In previous other studies, one research group reported that pyrrolnitrin had an ability to suppress of mycelial growth of F. graminearum. Other groups revealed that phenazine-1-carboxamide, a derivative of phenazine-1-carboxylic acid, could also inhibit the growth of F. graminearum and showed great potentials in the bioprotection of crops from FHB disease. In our recent work with Pseudomonas chlororaphis strain G05, however, we found that although the phz operon (phenazine biosynthetic gene cluster) was knocked out, the phenazine-deficient mutant G05Δphz still exhibited effective inhibition of the mycelial growth of some fungal phytopathogens in pathogen inhibition assay, especially including F. graminearum, Colletotrichum gloeosporioides, Botrytis cinerea. With our further investigations, including deletion and complementation of the prn operon (pyrrolnitrin biosynthetic gene cluster), purification and identification of fungal compounds, we first verified that not phenazines but pyrrolnitrin biosynthesized in P. chlororaphis G05 plays an essential role in growth suppression of F. graminearum and the bioprotection of cereal crops against FHB disease.

Reciprocal enhancement of gene expression between the phz and prn operon in Pseudomonas chlororaphis G05.

In previous studies with Pseudomonas chlororaphis G05, two operons (phzABCDEFG and prnABCD) were confirmed to respectively encode enzymes for biosynthesis of phenazine-1-carboxylic acid and pyrrolnitrin that mainly contributed to suppression of some fungal phytopathogens. Although some regulators were identified to govern their expression, it is not known how two operons coordinately interact. By constructing the phz- or/and prn- deletion mutants, we found that in comparison with the wild-type strain G05, phenazine-1-carboxylic acid production in the mutant G05Δprn obviously decreased in GA broth in the absence of prn, and pyrrolnitrin production in the mutant G05Δphz remarkably declined in the absence of phz. By generating the phzA and prnA transcriptional and translational fusions with a truncated lacZ on shuttle vector or on the chromosome, we found that expression of the phz or prn operon was correspondingly increased in the presence of the prn or phz operon at the post-transcriptional level, not at the transcriptional level. These results indicated that the presence of one operon would promote the expression of the other one operon between the phz and prn. This reciprocal enhancement would keep the strain G05 producing more different antifungal compounds coordinately and living better with growth suppression of other microorganisms.