Engineering of Yellow Mosaic Virus Resistance (YMVR) in Blackgram. Project ID: 1 April 2000 to 31 August Project Duration:

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1 Engineering of Yellow Mosaic Virus Resistance (YMVR) in Blackgram ID: Duration: Coordinator in Switzerland: PS1 1 April 2000 to 31 August 2004 Prof. Thomas Hohn University of Basel Botanisches Institut Schönbeinstrasse Basel, SWITZERLAND hohn@fmi.ch Coordinator in India: Prof. K. Veluthambi School of Biotechnology Madurai Kamaraj University Madurai , INDIA veluthambi@mrna.tn.nic.in RATIONALE Blackgram (Vigna mungo) is a major crop in India. Yellow mosaic disease, caused by vigna mungo yellow mosaic virus (VMYMV) is a major constraint in blackgram and also in mungbean cultivation. Since durable resistance has not been identified in any of the varieties of vigna mungo, generation of VMYMV resistance by genetic engineering has emerged as an important priority. To achieve resistance by genetic engineering appears to be the fastest and most promising approach. We have already cloned and sequenced the complete VMYMV DNA and the clone is infectious. Thus the viral control elements and protein coding sequences are available as tools and targets. We will follow three basic lines to obtain resistance: 1. Induced resistance. Enterotoxin genes, i.e. dianthin and barnase will be cloned under the control of the inducible rightward promoter of VMYMV. This promoter is induced by AC2, a product of the leftward promoter. The virus is expected to induce the engineered enterotoxin gene upon infection of plant containing the inducible transgene, cause death of the invaded cell and prevent virus spreading. 2. Silencing. Simultaneous presence of sense and antisense RNA sequences has been reported to repress expression from transgenes, endogenes and viral genes. Silencing can occur in the nucleus on the transcriptional level by promoter inactivation, e.g. by DNA methylation, and in the cytoplasm

2 on the posttranscritional level by RNA degradation. We want to induce both processes by transcribing VMYMV promoter sequences and coding regions in sense and antisense orientation. Transgenic plants allowing for both types simultaneously will be used to target both the nuclear and the cytoplasmic phase of virus replication. 3. Competitive inhibition. We want to analyse virus protein-protein interactions, determine the interactive domains, and construct genes for competitive inhibitors in transgenic plants on the basis of this information. SUMMARY OF THE ACHIEVMENTS OF THE FIRST PROGRAM PHASE adapted from the summary provided by the project partners To generate blackgram (Vigna mungo) resistance to Mungbean yellow mosaic virus-vigna (MYMV) by genetic engineering, three strategies were pursued: (i) RNA interference (RNAi) targeting both RNA and DNA forms of the virus; (ii) virus-induced cell death by using enterotoxin genes fused to inducible promoters; and (iii) competitive inhibition of protein-protein interactions between viral proteins and/or viral and cellular proteins. The main achievements are the following: A single Agrobacterium strain-based protocol for agroinfection of blackgram with the DNA A and DNA B partial dimers of MYMV was developed ( Madurai ). MYMV clones also caused yellow mosaic disease in mungbean (V. radiata) and mothbean (V. aconitifolia) but not in Nicotiana benthamiana and Nicotiana tabacum. With N. tabacum, a leafdisc assay was established in which MYMV was able to replicate following agroinoculation ( Madurai ). The MYMV clones were also infectious when delivered to blackgram plants biolistically via particle bombardment ( Basel ). A transient system to study the potency of RNAi against MYMV was developed, in which agroinfected blackgram seedlings are treated with anti-viral DNA constructs by particle bombardment ( Basel ). Several RNAi constructs were designed to express double-stranded (ds) RNA targeting both non-coding and coding regions of MYMV. Some of them were shown to be effective in the transient system: an artificial intron in the construct greatly improved the anti-viral effectiveness of RNAi ( Basel ). The two most potent RNAi constructs were subcloned into binary vectors with different selective markers, mobilised to agrobacterial strains and used for transformation of blackgram and N. tabacum ( Madurai ). Attempts to generate truly transgenic blackgram have not been successful so far ( Madurai ). In the case of N. tabacum, several transgenic plants carrying the RNAi construct targeting the MYMV promoter were obtained. However, none of them exhibited strong interference with MYMV replication in the leaf-disc assay ( Madurai ). Recovery of blackgram from MYMV-Vig infection was achieved by a nontransgenic approach using bombardment of infected plants with a

3 construct expressing dsrna. Although proving the feasibility of an RNAi approach to achieve virus resistance, such a method is economically costly and impractical. Therefore, the group looked for alternative possibilities how to introduce dsrna into infected plant cells. For this purpose, an RNaseIII-deficient E.coli strain capable of over-expressing dsrna was employed. Then, bacteria were constructed which express in the form of dsrna two segments of the MYMV-Vig genome (DNA A promoter and AC2 coding region), as well as the whole viral genome. To optimise parameters of plant treatments an additional bacterial strain was constructed to express dsrna cognate to a GFP gene. A model GFP transgenic plant, in which GFP silencing can be triggered by various means, was treated with the bacterial extract either by mechanical inoculation (involving gentle wounding of leaves with a plastic tip) or simple spraying. The latter treatment was not effective, whereas the former eventually resulted in systemic GFP silencing, thus demonstrating that RNAi could be induced by the dsrna-containing bacterial extracts. Treatments of blackgram seedlings were then undertaken to investigate efficacy of the anti-viral dsrna extracts. Mechanical treatment with the AC2 dsrna extract resulted in recovery of some of the blackgram seedlings from viral infection, while other extracts did not exhibit any effect. These results show that in principle a simple non-transgenic approach to achieve virus silencing and recovery from infection is possible, although further optimisation is required to achieve better antiviral effects of those extracts. To study the potency of dsrna expressed from a stably integrated transgene to target a geminivirus the group used a model easy-totransform N. benthamiana plant-african cassava mosaic virus (ACMV) system. Transgenic N. benthamiana plants carrying the 35S promoterdriven dsrna corresponding to the ACMV bi-directional promoter were produced and tested for resistance to ACMV infection. Initially, some of the inoculated plants developed viral symptoms. However, recovery from the infection could eventually be observed, which correlated with the reduction in viral titres (measured by semi-quantitative PCR) and the increase in viral DNA methylation (measured with methylation sensitive restriction enzymes followed by Southern-blot hybridization). These results suggest that a transgene locus expressing dsrna under the control of a strong constitutive promoter (such as the 35S promoter) may get self-silenced in plants, but that following viral infection the silencing may be suppressed (by the AC2 protein), eventually resulting in recovery from viral infection. This underscores the importance of the inducible promoters to be used in our anti-viral strategies. Promoters, transcripts and regulatory genes (AC1, coding for a replication protein, and AC2, coding for a transactivator of viral transcription) of MYMV were cloned and characterised both in infected blackgram plants (all the transcripts mapped) and in a transient expression system based on N. plumbaginifolia protoplasts (activities of all potential promoters and their regulation by AC1 and AC2 studied) (Basel). Promoters driving AV1 (coat protein) and BV1 (nuclear shuttle protein) genes, which exhibited lowest basal expression levels and strongest induction by AC2, were chosen for the virus-induced cell death approach ( Basel ). Transactivation of more than 30 plant genes by AC2 protein was

4 discovered using a transient expression system based on Arabidopsis protoplasts. Several corresponding promoters were cloned from the Arabidopsis genome and shown to be strongly inducible by AC2 from MYMV and a related African cassava mosaic begomovirus ( Basel ). Two of those promoters were chosen for the virus-induced cell death approach. The RNase barnase-barstar expression cassette with the MYMV bidirectional (AV2-AC1) promoter (Basel) and the dianthin expression cassette under the control of MYMV BV1 promoter ( Madurai ) were constructed to achieve the toxin gene (barnase or dianthin) expression in response to MYMV infection. These cassettes were integrated into binary vectors and mobilised to agrobacteria ( Madurai ). Transformation of blackgram is still in progress. Transformation of model tobacco plants with the barnase construct was not successful, probably due to leaky expression of the toxin gene ( Madurai ). Both barnase and diantin genes will be applied in the next phase of the project for a split gene approach, which should prevent any leaky expression of those genes in the absence of viral infection ( Basel ). Subcellular localization of MYMV proteins in plant protoplasts and potential viral protein-protein interactions in a yeast two hybrid system were studied ( Basel ). This information is useful for the competitive inhibition approach. The nuclear protein AC2 from MYMV was shown to play a dual role as a transactivator of transcription and a suppressor of silencing. Three mutant versions of this protein were constructed, in which both functions are abolished ( Basel ). The expression cassettes for those AC2 mutants were constructed and they will be used for the competitive inhibition approach in the next phase. Despite numerous efforts to transform blackgram with different binary vectors carrying different selective markers, most putative transgenic plants analysed at the molecular level so far turned to be Southernnegative. In the next phase of the project, the analysis of putative transgenics will be continued and optimised protocols of transformation will be applied. In particular, to increase transformation efficiency, a method of particle bombardement-mediated wounding of explants followed by incubation with agrobacteria will be used. Consistent with its function, AC2 was detected in nuclei of plant protoplasts (as visualized by GFP fusion) and its nuclear localisation signal (NLS) was mapped. Sub-cellular localization of other viral proteins was characterized in plant protoplasts. AC1 (replication associated protein) is localized in the nucleus, whereas AC4, whose function is unknown, associates with the cytoskeleton. All potential VMYMV proteins interactions were tested in the yeast twohybrid system. Preliminary results indicate that BV1, AV2, AC1 and AC2 interact with themselves. Consistently with its nuclear localization, AV1 interacts with importin a. Weak interaction of AV2 was observed with

5 AC2, AC3, AC4 and BC1. Co-workers in India: P.V. Shiva Prasad P. Lakshmi Kumari P. Thillai Chidambaram M. Saminathan R. Rajeswaran Remya Ramachandran Co-workers in Switzerland: Daniela Trinks Mikhael Rashid Akbergenov Pooggin