R. Srinivasan

Host-delivered RNAi-mediated root-knot nematode resistance in Arabidopsis by targeting splicing factor and integrase genes

Root-knot nematodes (RKNs) are one of the most important biotic factors limiting crop productivity in many crop plants. The major RKN control strategies include development of resistant cultivars, application of nematicides and crop rotation, but each has its own limitations. In recent years, RNA interference (RNAi) has become a powerful approach for developing nematode resistance. The two housekeeping genes, splicing factor and integrase, of Meloidogyne incognita were targeted for engineering nematode resistance using a host-delivered RNAi (HD-RNAi) approach. Splicing factor and integrase genes are essential for nematode development as they are involved in RNA metabolism. Stable homozygous transgenic Arabidopsis lines expressing dsRNA for both genes were generated. In RNAi lines of splicing factor gene, the number of galls, females and egg masses was reduced by 71.4, 74.5 and 86.6%, respectively, as compared with the empty vector controls. Similarly, in RNAi lines of the integrase gene, the number of galls, females and egg masses was reduced up to 59.5, 66.8 and 63.4%, respectively, compared with the empty vector controls. Expression analysis revealed a reduction in mRNA abundance of both targeted genes in female nematodes feeding on transgenic plants expressing dsRNA constructs. The silencing of housekeeping genes in the nematodes through HD-RNAi significantly reduced root-knot nematode infectivity and suggests that they will be useful in developing RKN resistance in crop plants.

Identification, validation and utilization of novel nematode-responsive root-specific promoters in Arabidopsis for inducing host-delivered RNAi mediated root-knot nematode resistance

The root-knot nematode (RKN), Meloidogyne incognita, is an obligate, sedentary endoparasite that infects a large number of crops and severely affects productivity. The commonly used nematode control strategies have their own limitations. Of late, RNA interference (RNAi) has become a popular approach for the development of nematode resistance in plants. Transgenic crops capable of expressing dsRNAs, specifically in roots for disrupting the parasitic process, offer an effective and efficient means of producing resistant crops. We identified nematode-responsive and root-specific (NRRS) promoters by using microarray data from the public domain and known conserved cis-elements. A set of 51 NRRS genes was identified which was narrowed down further on the basis of presence of cis-elements combined with minimal expression in the absence of nematode infection. The comparative analysis of promoters from the enriched NRRS set, along with earlier reported nematode-responsive genes, led to the identification of specific cis-elements. The promoters of two candidate genes were used to generate transgenic plants harboring promoter GUS constructs and tested in planta against nematodes. Both promoters showed preferential expression upon nematode infection, exclusively in the root in one and galls in the other. One of these NRRS promoters was used to drive the expression of splicing factor, a nematode-specific gene, for generating host-delivered RNAi-mediated nematode-resistant plants. Transgenic lines expressing dsRNA of splicing factor under the NRRS promoter exhibited upto a 32% reduction in number of galls compared to control plants.

Regulatory sequences of the Arabidopsis thaliana Rps19 , a nuclear gene encoding mitochondrial ribosomal protein subunit, extend into the upstream gene

Genes encoding subunits of the mitochondrial ribosomal protein complex are distributed between the mitochondrial and nuclear genomes. In Arabidopsis thaliana only seven out of the nearly 70 genes coding for mitochondrial ribosomal subunit proteins are present in the mitochondrial genome. Nevertheless, these genes are co-ordinately expressed. Here, we present the first report of characterization of promoter of a plant mitochondrial ribosomal protein gene AtRps19 (At5g47320), a single copy nuclear gene in A. thaliana. Analysis of transgenic A. thaliana plants carrying seven different AtRps19 upstream fragments linked to the uidA reporter gene revealed that the 879xa0bp fragment containing the 5′ UTR and the intergenic region is capable of driving gene expression in most of the vegetative tissues. However, inclusion of 447xa0bp of the upstream gene sequences was essential for obtaining full expression in all tissues including anthers and pollen. Thus, we provide the first experimental proof of overlapping genes in plants. qRT-PCR analysis showed that uidA and native AtRps19 transcript levels were comparable in plants carrying the D4 (−676/+650) construct, indicating that the 1326xa0bp D4 fragment represents the native promoter of the AtRps19 gene. Comparison of AtRps19 promoter with the widely used CaMV35S promoter showed that AtRps19 promoter is capable of driving gene expression in all tissues including anthers and pollen but was less efficient than the CaMV35S promoter.

Characterization of root-knot nematode responsive and root-specific promoter containing PIN domain from Arabidopsis thaliana (L.) Heynh

Root-knot nematodes (Meloidogyne incognita) are obligate plant parasites, causing significant economic loss, that alter expression of host genes in order to establish and maintain their feeding sites in the roots of host plants. In the present study, a nematode-responsive-root-specific gene (AT1G26530) was identified which expressed in roots of Arabidopsis thaliana after nematode infection. Quantitative RT-PCR analysis of this gene revealed maximum (~2.58 fold) up-regulation at 21 days post inoculation of nematode. A 1580 bp region upstream of the translational start site of AT1G26530 was isolated and transformed into Arabidopsis through floral dip method. On analysis of Arabidopsis transgenic plants harboring AT1G26530 prm:: GUS fusion, reporter gene expression was seen exclusively in galls after nematode inoculation. Interestingly, strong GUS activity was observed at early stages of nematode infection, starting from 14 days and was sustained up to 30 days post inoculation. Furthermore, 85 to 93% galls exhibited GUS activity in the nematode feeding sites. The specificity of the activity of the AT1G26530 promoter, in terms of nematode-responsiveness and rootspecificity, makes it a suitable candidate to express dsRNA of nematode genes and engineer plants with resistance against root-knot nematodes using HD-RNAi technology.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants

A promoter trap mutant line of Arabidopsis carrying a promoterless β-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5 and 3 promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

The TRAF Mediated Gametogenesis Progression (TRAMGaP) Gene Is Required for Megaspore Mother Cell Specification and Gametophyte Development

TRAMGaP gene coordinates the expression of diverse sets of genes to modulate MMC specification and gametophyte development in Arabidopsis. In plants, the role of TRAF-like proteins with meprin and the TRAF homology (MATH) domain is far from clear. In animals, these proteins serve as adapter molecules to mediate signal transduction from Tumor Necrosis Factor Receptor to downstream effector molecules. A seed-sterile mutant with a disrupted TRAF-like gene (At5g26290) exhibiting aberrant gametogenesis led us to investigate the developmental role of this gene in Arabidopsis (Arabidopsis thaliana). The mutation was semidominant and resulted in pleiotropic phenotypes with such features as short siliques with fewer ovules, pollen and seed sterility, altered Megaspore Mother Cell (MMC) specification, and delayed programmed cell death in megaspores and the tapetum, features that overlapped those in other well-characterized mutants. Seed sterility and reduced transmission frequency of the mutant alleles pointed to a dual role, sporophytic and gametophytic, for the gene on the male side. The mutant also showed altered expression of various genes involved in such cellular and developmental pathways as regulation of transcription, biosynthesis and transport of lipids, hormone-mediated signaling, and gametophyte development. The diverse phenotypes of the mutant and the altered expression of key genes related to gametophyte and seed development could be explained based on the functional similarly between At5g26290 and MATH-BTB domain proteins that modulate gene expression through the ubiquitin-mediated proteasome system. These results show a novel link between a TRAF-like gene and reproductive development in plants.

Promoter Trapping and Deletion Analysis Show Arabidopsis thaliana APETALA2 Gene Promoter Is Bidirectional and Functions as a Pollen- and Ovule-Specific Promoter in the Reverse Orientation

The Arabidopsis thaliana promoter trap mutant Bitrap-112 expressing green fluorescent protein (GFP) gene in the ovules was found to carry transferred DNA (T-DNA) insertion at −309 position of the APETALA2 (AP2) gene. Bitrap-112 line did not show phenotype associated with the AP2 mutation, suggesting that T-DNA insertion did not interrupt the AP2 promoter. Further, head-to-head orientation of GFP and AP2 genes indicated that the AP2 promoter could be bidirectional. A detailed deletion analysis of the upstream sequences of the AP2 gene was done to identify the promoter. GUS assay of transgenic A. thaliana plants carrying various AP2 upstream fragments fused to the uidA gene showed that ~200-bp 5′ UTR sequences are capable of driving gene expression at low levels in vegetative tissues whereas inclusion of further upstream sequences (~300xa0bp) enhanced uidA expression comparable to native AP2 expression levels in various tissues including ovules. In the reverse orientation, the 519-bp AP2 upstream fragment was found to drive gene expression in immature ovules and pollen. Absence of antisense transcripts corresponding to the sequences upstream of AP2 gene in wild-type A. thaliana plants suggests that promoter trapping has uncovered a cryptic promoter, which in reverse orientation is capable of driving gene expression in ovules and anthers.