"Geminiviruses" are an emerging family of plant viruses of increasing economic impact. Tomato leaf curl virus (TLCV) is the causal agent of a severe disease of tomato in northern regions of Australia. The progressive southward spread of this pathogen threatens the major tomato-growing regions of Queensland. No effective forms of natural or transgenic resistance are available. Geminiviruses are also affecting cotton, tomato and other food crops in Pakistan and are causing impact of national magnitude on the country's economy. The focus of this project is to characterize the main geminiviruses infecting cotton and tomato in Pakistan and Australia, and to develop geminivirus resistant varieties of these crops.
The collaborating research institutions are National Institute for Biotechnology and Genetic Engineering (NIBGE) in Pakistan and Asian Vegetable Research and Development Centre (AVDRC) in Taiwan.
Field surveys in Pakistan resulted in the identification of geminiviruses infecting cotton, tomato, okra, tobacco and chilies. Full-length clones of corresponding DNA A of these viruses have been obtained from infected plants and their characterization is in progress. Cotton leaf curl disease is caused by a virus complex that is transmitted by whiteflies. Several distinct begomoviruses have been cloned from infected cotton plants from Pakistan. However, infectious clones of CLCuV alone are unable to induce typical disease symptoms. A single species of a DNA satellite called DNA is essentially required for the induction of disease symptoms. DNA appears to be driving evolution of the disease that is able to capture several distinct begomoviruses for replication and encapsidation. DNA shares some of the features of a geminivirus satellite DNA first reported in Australia.
In Australia, genome characterization of TLCV is well advanced. The recent field surveys of TLCV in Darwin and a remote region of Queensland resulted in the identification of strain diversity and compiling a distribution map for the virus. TLCV frequently contains defective DNA molecules known as DI DNAs which are approximately 1/4 or 1/2 the size of viral genome. These are encapsidated and supported for replication by TLCV. Work has been initiated to test gene constructs based on the DI DNA for virus inhibition.
The information generated in this phase of the project will form the basis for designing synthetic gene constructs and devising detection tools for field surveys.

Field surveys in Pakistan resulted in the identification of a new variant of the Cotton leaf curl virus (CLCuV) from Burewala area in Punjab. This variant appears to have overcome resistance, both in commercial varieties and in sources of resistance used for breeding. The surveys showed that Burewala strain has reached up to Vehari area but the major cotton belt including Multan which is in the close vicinity of Burewala is as yet unaffected. Full-length begomovirus DNA A was amplified from Burewala and other sites in Vehari district. CLCuV DNA A alone is unable to induce typical disease symptoms. A single species of a DNA satellite called DNA b is essential for the induction of disease symptoms. Complete nucleotide sequence of DNA b of Burewala strain of CLCuV was determined and found to be 92% homologous with strains reported previously. The resistance breaking CLCuV isolate is being characterized in an effort to understand the basis of this increased pathogenicity.
Surveys in Pakistan resulted in the identification of geminiviruses infecting other crops including chili, tomato, cucurbits, and papaya. Surveys show prevalence of a bipartite begomovirus on chili crops and multiple infections of monopartite begomoviruses that require DNA b for symptom induction. Previous surveys in Pakistan showed the presence of several distinct begomoviruses in tomato. The surveys indicated that tomato plants in Pakistan were infected with tomato leaf curl virus (TLCV) from Banglore. This virus produces disease symptoms in the absence of DNA b. These surveys also showed widespread infection of tomato leaf curl virus on cucurbits and distribution of Indian mungbean yellow mosaic virus in Pakistan indicating that geminiviruses are a wide spread problems of increasing economic impact in Pakistan.
Field surveys of TLCV in northern Australia resulted in the identification of TLCV strains. Sequence analysis of 11 TLCV field isolates revealed a close relationship between the isolates. The newly introduced silverleaf whitefly, Bemisia tabaci biotype B, was found to form high population densities for the first time in Mareoba, approximately 150 km from the nearest known TLCV infected area. The viral host range was found to be narrow and of 58 species of crop plants and weeds inoculated using the B biotype of whiteflies, only 11 became infected, including five that did not show foliar symptoms.
To allow biological assays in the laboratories, an infectious clone of CLCuV was tested on cotton, datura, Nicotiana bentamiana and N. tabacum. Mixed inoculation of CLCuV DNA and DNA b was found to produce disease symptoms using biolistic inoculation. Efforts to infect cotton by agroinaculataion were unsuccessful. We also found that the replication of CLCuV DNA b is supported by Australian isolate of TLCV. Mutational analysis to characterize the role of DNA b in CLCuV infection and symptom expression is in the progress.
TLCV infectivity is usually associated with production of subgenomic DNA known as defective-interfering DNA molecules (DI DNAs). DI DNAs are encapsidated and supported for replication by TLCV. A region of approximately 300 nucleotides is conserved in all TLCV DI DNAs studied so far. The role of this conserved region in viral replication and/or encapsidation is unknown. Mutational analysis of infectious clones of a DI DNA to determine the role this region is in progress. This study is aimed of developing resistance strategies based on the use of DI DNAs.
Suppression of DNA b in transgenic Nicotiana tabacum was shown to protect plants against cotton leaf curl disease. The gene construct of complementary ORFs of DNA b was cloned in binary vectors and was transformed in Nicotiana tabacum. The transgenic plants showed no disease symptoms when infected by CLCuV either through whiteflies or by agroinfiltration. Resistance constructs based on the viral genome were also produced. These were based on RNA silencing of the genes for the coat protein and for the replication associated protein. The efficiency of these constructs will be checked in transient assays and then transformed in tobacco and cotton.
Post-transcriptional gene silencing (PTGS), thought to be a defence mechanism against viruses, can be induced in plant by transforming them with intron-spliced inverted-repeat construct. Several transgenic tomato lines with the TLCV C2 hairpin construct were produced in Australia. However, only one line (C2-B) showed delayed and attenuated symptoms after TLCV inoculation. Strong silencing signal (siRNA) was detected in the transgenic plants both before and after inoculation. This suggests post-transcriptional gene silencing was induced in this transgenic line.

Field surveys of geminiviruses in Australia and Pakistan
Tomato leaf curl virus (TLCV) in Australia has now spread to Mossman where more intense vegetation, a high population of silverleaf whiteflies and the volume of traffic in the area is likely to contribute to further spread of TLCV to the major tomato growing regions.
A resistance breaking variant of the cotton leaf curl virus (CLCuV) in Pakistan has spread to several districts in the Punjab province. Analysis of viruses involved showed that, in addition to a geminivirus, a small satellite DNA (ssDNA) is present. Similar circular ssDNA satellites have recently been isolated from plants infected with other geminiviruses. The satellites, named DNA-alpha, depend on the helper viruses for their proliferation and, in turn, induce disease symptoms. The DNA associated with the resistance breaking variant is a recombinant of two DNA molecules isolated previously from cotton and tomato. Plants infected with the resistance breaking virus also contained recombinant of CLCuV and Okra yellow vein mosaic virus. Thus, viral recombination and mobilization of new viral components appears to be associated with a new epidemic of cotton leaf curl disease (CLCuD).
In other crops, CLCuV with a distinct DNA and tomato leaf curl New Delhi virus (ToLCNDV) were detected in chillies. ToLCNDV also causes widespread infection on cucurbits including squash, melon, muskmelon and watermelon. A severe disorder of melon was observed in the Punjab that was caused by a geminivirus/DNA alpha complex. Sequence analysis of DNAs isolated from Ageratum showing yellow vein disease indicated that CLCuV DNA and the DNA alpha of Ageratum yellow vein virus cause this disease.
Viral genome characterisation
Sequences analysis of 28 DNA alpha molecules revealed a highly conserved genome organisation. Two major groups of DNA alpha satellites were identified, one within the Malvaceae hosts and the second from plants within the Solanaceae and Compositae. The DNA sequence responsible for inducing disease symptoms was confirmed. Further analysis of RNA from the transgenic plants identified one amino acid protein, responsible for symptom development.
Development of virus-resistant plants through genetic engineering:
RNA interference (RNAi) is a powerful tool for controlling gene expression. To induce RNA silencing against TLCV we transformed tomato plants with an intron-spliced hairpin sequence from TLCV and obtained transgenic lines producing high levels of small interfering RNA that is a hallmark of RNAi. Inoculation of these plants with TLCV resulted in attenuated infection.
Previously we showed that suppression of DNA alpha in transgenic Nicotiana tabacum protected plants against CLCuD. New constructs for simultaneous silencing of the viral DNA and DNA beta were tested in transient assay and were found to be effective. These constructs are being transformed in cotton and tobacco. RNAi constructs for ToLCNDV are being transformed in tomato. We also targeted the silencing of the CLCuV replication-associated protein as well as virulence determinant encoded by DNA and found these transgenes protect the plants from infection.
About 300 cotton genotypes and breeding lines at Vehari Research Station were analysed for resistance against CLCuV. A cotton genotype was found to remain completely virus free. In field trials no plants of transgenic cotton Coker-312 were infected with CLCuD while 100% untransformed control plants were infected. These results indicate that resistance in these lines is stably inherited. Seeds collected from these plants are being used for further field studies.

Field surveys of geminiviruses:

Cotton leaf curl disease (CLCuD) is a major constraint to the production of cotton in Pakistan. The disease is caused by a combination of a DNA ' and either Cotton leaf curl Alabad virus, Cotton leaf curl Multan virus (CLCuMV), Cotton leaf curl Khokhran virus (CLCuKV) or Papaya leaf curl virus. The DNA ' is responsible for disease symptoms and depends on CLCuV for replication and encapsidation.

Field surveys indicated the resistance breaking variants of CLCuV were spreading in the Punjab. A recombinant DNA A with sequences derived from CLCuMV and CLCuKV was found to be associated with resistance breakdown. A diverse number of DNA was also isolated and sequenced.

A severe epidemic of tomato leaf curl disease has appeared in tomato crops in the Sindh province. Both ToLCNDV and Tomato leaf curl Bangalore virus and a DNA were found in diseased samples. Surveys of other crops revealed ToLCNDV, Zucchini yellow mosaic virus and ChLCMV with recombinant DNA s in chilies, melon, Ageratum conyzoides and papaya.

Viral genome characterization:

We have already showed that a complementary-sense ORF of CLCuV DNA ', 'C1, is required for inducing disease symptoms. The expression of the 'C1 ORF from a Potato virus X (PVX) vector developed symptoms of CLCuD in tobacco confirming that the 'C1 is involved in pathogenicity. The function of 'C1 protein in virus movement was investigated. Tomato plants co-inoculated with a movement deficient component A of TLCNDV and CLCuV DNA ' exhibited severe symptoms and contained replicative forms of both DNA A and DNA ' in the newly developing leaves. The control tomato plants inoculated with DNA A alone did not show any symptoms and Southern blot analysis failed to detect the virus. Transient expression of DNA '/GFP fusion protein in onion and Nicotiana benthamiana indicating that the 'C1 localized to the cell periphery in epidermal cells of these plants. Together, these results indicate that 'C1 plays a role in systemic spread of the virus. The ability of DNA ' to be supported for replication by a number of geminiviruses combined with its ability to spread a movement deficient virus component is probably contributing to the emergence of new diseases in tropical and sub-tropical crops.

The role of nuclear shuttle protein (NSP) and movement protein (MP) genes in pathogenecity of ToLCNDV was investigated. No phenotypic changes were observed when MP was expressed through a PVX vector and under the Cauliflower mosaic virus (CaMV) 35S promoter. The expression of NSP from PVX developed downward leaf curling in N. benthamiana and hypersensitive response (HR) in N. tabacum and tomato. This suggests NSP of ToLCNDV is a pathogenicity determinant. Mutagenesis analysis showed that the N-terminal amino acids of NSP are important for the hypersensitive response in tobacco. To study the role of NSP in the suppression of gene silencing, several gene constructs in fusion with GFP have been produced.

Development of virus-resistant plants through genetic engineering:

Transgenic tobacco plants transformed with an RNAi construct of CLCUV targeting either complementary-sense genes (AC1-AC4) or both DNA A and DNA ' were resistant to infection by the homologous virus. Transgenic plants were also exposed to field isolates of CLCuD by whitefly inoculation. Two out of ten lines developed disease symptoms. The geminivirus in these infected plants was identified as ChLCMV and DNA ' of CLCuV or chili beta. These results indicate that induced resistance may be virus-specific.

A cotton genotype (NIBGE-115), that was found to be completely virus free when inoculated with the resistance breaking strain, was multiplied and is being used as a source of resistance in the breeding program at NIBGE. Cotton lines transformed with silencing constructs targeting DNA A sequences (AC1-AC4) have shown promising results in field trials at Vehari station where breakdown of natural resistance is a major problem. An RNAi construct targeting AC1 and 'C1 simultaneously has been transformed in cotton and fifteen plants were regenerated from various batches of hypocotyls inoculation experiments. These lines will be tested for virus resistance at the F1 stage.

Final Report received

Final Report received