Downstream promoter sequence of an Indian isolate of Rice tungro bacilliform virus alters tissue-specific expression in host rice and acts differentially in heterologous system

Abstract

An Indian isolate of Rice tungro bacilliform virus from West Bengal (RTBV-WB) showed significant nucleotide differences in its putative promoter region when compared with a previously characterized isolate from Philippines. The transcription start site of RTBV-WB was mapped followed by assessing the activity and tissue-specificity of the full-length (FL) promoter (-231 to +645) and several of its upstream and downstream deletions by studying the expression of beta-Glucuronidase (GUS) reporter gene in transgenic rice (Oryza sativa L. subsp. indica) plants at various stages of development. In addition to the expected vascular-specific expression pattern, studied by histochemical staining, GUS enzymatic assay and northern and RT-PCR analysis, two novel patterns were revealed in some of the downstream deleted versions; a non-expressing type, representing no expression at any stage in any tissue and constitutive type, representing constitutive expression at all stages in most tissues. This indicated the presence of previously unreported positive and negative cis-regulatory elements in the downstream region. The negative element and a putative enhancer region in the upstream region specifically bound to rice nuclear proteins in vitro. The FL and its deletion derivatives were also active in heterologous systems like tobacco (Nicotiana tabacum) and wheat (Triticum durum). Expression patterns in tobacco were different from those observed in rice suggesting the importance of upstream elements in those systems and host-specific regulation of the promoter in diverse organisms. Thus, the RTBV-WB FL promoter and its derivatives contain an array of cis-elements, which control constitutive or tissue- and development-specific gene expression in a combinatorial fashion.

Virus-induced gene silencing in rice using a vector derived from a DNA virus

“…The promoter of the Indian isolate of RTBV when expressed transgenically is active mainly in the vascular bundles in a mature rice plant (Mathur and Dasgupta 2007). We reasoned that the use of a constitutive promoter, rather than a tissue-specific one, would be more appropriate for a VIGS vector.…”

“…Hence, RF2a-and RF2b-overexpressing transgenic rice lines do not show symptoms upon RTBV inoculation (Dai et al 2008). Though the Box II element is not completely conserved in the two groups of RTBV, from Philippines and India (Mathur and Dasgupta 2007), an interaction of host factors with elements resembling Box II in the RTBV-WB promoter, causing the tungro symptoms, cannot be ruled out. In pRTBV-MVIGS, since RTBV promoter is replaced by MUP, the lack of Box II element or other similar elements, otherwise interacting with RF2a and RF2b, may contribute to the lack of symptoms in plants upon agroinoculation with pRTBV-MVIGS.…”

“…The transcription start site in pRTBV203, a cloned DNA representing an Indian RTBV isolate, has been mapped at position 7392 (Mathur and Dasgupta 2007). To construct pRTBV-Inf, an approximately 3.9-kb fragment corresponding to position between 6955-7934 and 1-2930 of pRTBV203 was amplified using the Expand Long Template PCR System (Roche, Mannheim, Germany) using primer P1 (Table S1) and standard M13 reverse primer, using pRTBV203 as the template.…”

Bacilliform DNA‐containing plant viruses in the tropics: commonalities within a genetically diverse group

“…At present, 12 complete sequences of RTBV are available in the database, eight of which are from South‐East Asia and four from India. The genome sequences fall into two distinct groups, the South Asian and South‐East Asian groups, sharing approximately 75% sequence identity, the nucleotide identities within a group being about 95% (Fan et al ., ; Mathur and Dasgupta, , ; Nath et al ., ).…”

Identification of Leaf Promoters for Use in Transgenic Wheat

“…Currently, the maize ubiquitin 1 [ 35 ] and the rice actin 1 constitutive promoters [ 36 ] are frequently used to direct expression of transgenes in wheat. Additionally, the semi-constitutive rice tungro virus promoter [ 37 , 38 ] has been used to express genes in the aerial parts of the wheat plant [ 9 ]. More recently, the rubisco small subunit (Rubisco) gene promoter from wheat was shown to direct expression in immature wheat embryos and tobacco leaves in transient assays [ 39 ].…”