Molecular studies on a complex of potyviruses infecting solanaceous crops, and some specific virus-host interactions

This thesis constitutes a comprehensive analysis of the molecular and biological characteristics of three potyviruses (genus Potyvirus, family Potyviridae) naturally occurring in cultivated and wild species of family Solanaceae: Peru tomato virus (PTV), Potato virus V (PVV) and Wild potato mosaic virus (WPMV). In addition, the studies presented in this thesis focus on the genetic variability of isolates of PTV and PVV and on the role of the Potato virus A (PVA) 6K2 protein as a host-specific determinant of virus movement and symptom induction. Determination of the complete genomic sequences of PVV, PTV and WPMV demonstrated that these viruses are typical members of the genus Potyvirus. Furthermore, comparison of the polyprotein amino acid sequences and the biological and serological characteristics of these three viruses supported their current taxonomic position as independent species of the genus Potyvirus. The nucleotide sequences of the P1 protein, coat protein and non-translated regions of European and South American PVV isolates were determined and compared. Results showed limited genetic variability among the European isolates, in contrast to the higher variability found among the South American isolates of PVV. Phylogenetic analysis defined two distinct clusters, grouping the European isolates together but placing two South American isolates to a different group; these two isolates of PVV did not induce a hypersensitive response in an Nv gene-carrying potato cultivar in contrast to the European PVV isolates. Thus, European and South American PVV isolates belong to different strain groups. In addition, great genetic variability was detected among PTV isolates. Analysis of phylogenetic relationships among PTV, PVV, WPMV and other members of the genus Potyvirus commonly found infecting solanaceous crop plants showed that PTV, PVV and WPMV are the most closely related viruses which together with Potato virus Y, Pepper mottle virus, Pepper severe mosaic virus and Pepper yellow mosaic virus constitute a group distinguishable from other potyviruses. Thus, members of this group seem to share a common ancestor. The 6K2 protein of PVA was modified by deleting various portions or by introducing six histidine residues (6xHis) into various positions of this protein. These modifications disturbed functions required for viral infection in Nicotiana tabacum. Furthermore, inoculation of the insertion constructs to N. benthamiana plants did not result in systemic infection with the exception of one plant. This plant lacked typical PVA symptoms but had virus titers similar to the plants infected with the wild type virus: a single point mutation (Gly2 ® Cys2) in the 6xHis-containing 6K2 had restored the viral movement functions. However, partial deletion of the 6xHis-tag to gain the original size of the 6K2 protein was required to restore the induction of symptoms in N. benthamiana and to enable systemic infection of N. tabacum. Taken together, these results indicate the 6K2 is a host-specific determinant for long-distance movement and exemplify that mutations that arise during viral propagation represent a mechanisms by which viruses can evolve and adapt to different hosts

Transgenic resistance to PMTV and PVA provides novel insights to viral long-distance movement

The studies in this thesis describe forms of transgenic resistance to plant viruses and how they can be used for studying viral infection cycle. S. tuberosum cv. Saturna expressing the CP gene of Potato mop-top virus (PMTV) was grown in a field infested with the viruliferous vector of PMTV, S. subterranea. The incidence of PMTV-infected tubers was lower in the CP-transgenic potato than in non-transgenic potato. RNA dot-blot analysis revealed that in tubers infected with PMTV, all three RNAs were present. N. benthamiana plants expressing the CP gene of PMTV were inoculated by two different methods i) mechanical inoculation to leaves and ii) growing plants in soil infested with viruliferous S. subterranea. Results showed that the expression of the transgene-derived RNA (or CP) inhibits replication of homologous RNA 2 in transgenic N. benthamiana. Furthermore, the results showed that transgene-mediated resistance to PMTV differs in roots and leaves. Mechanical inoculation with PMTV on CP-transgenic N. benthamiana resulted in symptomless, systemic movement of RNA 1 and RNA 3, but not the CP-encoding RNA (RNA 2). These findings show that PMTV RNA 1 and RNA 3 can infect and move systemically in N. benthamiana without the CP and RNA 2. N. benthamiana transformed with the P1 and VPg cistron, respectively, of Potato virus A (PVA) displayed: i) resistance to PVA infection, ii) susceptibility, or iii) systemic infection followed by recovery from PVA infection of new leaves. Long-distance transport of PVA from lower, infected parts of recovered plants was compromised in the recovered tissue. This result suggests that PVA is moving as ribonucleoprotein complex other than virus particles. N. benthamiana transformed with a polycistronic transgene encoding the CI-NIa-CP cistrons of PVA was susceptible to PVA infection. VPg (the N-proximal part of NIa) is a well-known virulence factor of potyviruses and its possible role in suppression of RNA silencing was studied. PVA VPg was found to increase the severity of disease symptoms when expressed from a Potato virus X vector in N. benthamiana. However, PVA VPg did not show apparent RNA silencing suppression activity. The reason why the polycistronic transgene did not provide resistance could not be resolved

Biological and molecular studies on potatovirus interactions using Potato virus S and Potato virus Y as model systems

Potato (Solanum tuberosum L.) is a staple food in the world. Potato virus Y (PVY; Potyvirus: Potyviridae) is an important virus that affects yield and quality of potatoes. PVY exists as biologically distinct strains: PVY-N induces systemic veinal necrosis in tobacco, PVY-O causes hypersensitive response (HR) in potato cultivars carrying the Ny gene and PVY-NTN produces necrotic rings on the tubers of sensitive potato cultivars. The vsiRNA profiles of three distinct PVY strains, ordinary (PVY-O), tobacco-veinal necrotic (PVY-N) and tuber-necrotic (PVY-NTN) strains were determined in potato cv. Russet Burbank. The frequency and distribution of vsiRNAs varied among different strains. PVY-NTN infected plants accumulated the highest population of PVY-vsiRNAs in comparison to plants infected with PVY-O and PVY-N. In PVY-infected plants, the 21 nt class was predominant whereas in healthy potato plants 24 nt class had the maximum population. VsiRNAs were derived from every nucleotide position of the PVY genome and certain hotspots were identified which produced relatively more vsiRNAs. Additionally, six novel microRNAs were found in PVY-infected plants. Potato virus S (PVS; Carlavirus: Betaflexiviridae) is another important potato virus distributed worldwide. The outcome of mixed infection of PVS and PVY was studied under controlled conditions in three potato cultivars, Defender, Desiree and Russet Burbank. Results showed that PVS has an antagonistic effect on PVY replication in mixed infection. The antagonistic effect was associated with less severe symptoms in dual infections as well as reduced PVY multiplication. Symptoms of PVS were not visible in Desiree and Russet Burbank plants except Defender which showed bronzing spots on the leaves. PVY symptoms included mosaic, mottling and leaf drop. It was found that the antagonistic effect of PVS on the replication of PVY is independent of host genetic background as the same pattern was found in all three potato cultivars. Potato cultivars such as Desiree carrying the Ny gene show HR to infection with the ordinary strain of PVY. In comparison to Russet Burbank, PVY levels in Desiree decreased with increasing number of days post-inoculation. The HR was found to be specific to PVY-O and was not elicited by infection by PVY-N or PVY-NTN

A Review Paper on Potato Virus Y (PVY) Biology, Economic Importance and its Managements

PVY is distributed worldwide in potato growing areas, tobacco growing areas and outdoor crops of tomato and pepper in warm countries. The different strains of PVY develops different symptoms on different parts of the host plant species. The PVY have a host range of  495 species in 72 genera of 31 families. Potato isolates have historically been divided into three main strain groups. The different strains of PVY have different stability under thermal inactivation point, dilution end-point and longevity in vitro. PVY have ssRNA nucleotide and two proteins (VPg and coat protein). The genomic RNA of PVY is positive sense and approximately 9700 kb in length excluding the poly(A) tail. PVY is spread from plant to plant either by aphids, mechanical means or by contact. In the family Aphidinae, Myzus persicae is clearly the most important vector of PVY given that it is widespread and with high transmission efficiency. Potato  virus  Y  belongs  to  a  group  of  the  most  important  potato  viruses  infecting  the  potato, tobacco, pepper and tomato. PVY is the most important viral pathogen in potato worldwide and can cause yield loss of 10-100% and 39-75% on tobacco. The two main types of resistance in potato are extreme  resistance  and hyper-sensitive resistance. The generation  of  resistant  cultivars  is  considered  the most  economic  and  environmentally  acceptable  way  of  controlling  viral  diseases  in  potato. Vector control plays an important role in management of PVY. Controlling the aphid plays a great role in the management of Potato virus Y. Since PVY is among the most important viral diseases of potato and that cause significant yield loss, so understanding its biology and developing an efficient management strategy is very important.  In the real world there is no singly effective management of several important diseases, on behalf of this, searching a good integrated management approach is very crucial. Keywords: Coat Protein; Hyper-sensitive Resistance; Myzus persicae; Potato; ssRNA; Vector control

Molecular basis of resistance to soybean mosaic virus: reverse transcription (RT)-PCR and strain complementation analysis in soybeans

Soybean (Glycine max(L.) Merr.) is grown in both developed and developing countries primarily for vegetable oil and protein products. It is the third most widely planted crop in the United States. Soybean mosaic virus (SMV) can affect soybean production by causing reduction in grain yield and quality. The virus is a member of the large and economically important family Potyviridae and several strains have been identified. Soybean lines exist that contain single genes (Rsv) for resistance to SMV. Therefore, SMV and soybean have been used as a model to understand plant resistance to virus disease. One facet of this study has been development of an accurate reverse transcription-polymerase chain reaction (RT-PCR)-based assay for differentiation of SMV strains. This technique was then used to study evidence for strain complementation in soybeans;Because SMV strains can occur as mixtures in infected soybean (cultivar Williams 82), an RT-PCR was developed to detect and discriminate among strains at the RNA level. The assay was applied to virus strains G2 and G7 and utilized oligonucleotides complementary to unique regions in the cylindrical inclusion protein cistron. Total RNAs from plants infected with either strain or a mixture of strains were amplified with G2 and G7 specific primers. Specific fragments were obtained. The assay allowed discrimination of strains in a mixed infection of a single soybean host plant;Previous evidence for putative complementation of the SMV strain G2, that does not infect soybean lines containing the Rsv gene, was reexamined. The model suggested that cell-to-cell spread of G2 could occur in plants systemicaly preinfected with the related SMV G7 or G7a strains in soybean lines PI 96983, L78-379, and Davis (immune to G2, but susceptible to G7 and G7a). Time course assay revealed differences in infectivity profiles of tissues from inoculated lines PI 96983 and L78-379. The optimum time for analysis of virus strains in the infected plants of the two genotypes was 20 to 25 days after inoculation. Analysis provided no evidence that G2 movement could be complemented by G7 or G7a in lines PI 96983, L78-379 and Davis

Identification of two potyviruses of phaseolus vulgaris in South Africa

Summary in English.Bibliography: pages 105-125.A survey was conducted by researchers at ARC-PPRI on dry beans (Phaseolus vulgaris) during 1993. All the viruses known to occur on dry beans in South Africa were found, as well as a few unidentified viruses. Of these, samples 93/1 and 93/65 form the basis of this thesis. Electron microscopy (EM) indicated that these viruses could be potyviruses, as they were flexuous particles of approximately 700 to 800 nm. Observation of pinwheels in ultrathin sections of Nicotiana benthamiana infected with isolate 93/1 and Phaseolus vulgaris infected with isolate 93/65, confirmed that the viruses probably belonged to the Potyvirus genus, family Potyviridae. Further serological tests indicated that the viruses were related but not homologous to strains of clover yellow vein (CIYW) and blackeye cowpea mosaic (BICMV) viruses respectively. None of these viruses have previously been described as occurring in South Africa. As we were unable to positively identify the viruses with serological methods, we needed to characterise these viruses on a molecular level. Potyvirus specific oligonucleotide primers were used for PCR amplification of viral eDNA The primers amplify an approximately 700 bp fragment of the virus genome, spanning the 3' noncoding region as well as a part of the coat protein gene: one primer is complementary to the poly(A) tail, and the other to a sequence coding for a conserved block of amino acid sequences (also known as the WCIEN block) in the mid-region of the coat protein. The nucleic acid sequences of the PCR products were compared to that of other potyviruses to positively identify these isolates

Differential pathogenicity of two different recombinant PVYNTN isolates in Physalis floridana is likely determined by the coat protein gene

A previous study has identified two types of recombinant variants of Potato virus Y strain NTN (PVYNTN) in China and sequenced the complete genome of the variant PVYNTN-HN2. In this study, the complete genome of isolate PVYNTN-HN1 was fully sequenced and analyzed. The most striking difference between the two variants was the location of recombinant joint three (RJ3). In PVYNTN-HN1, like other typical European-PVYNTN isolates such as PVYNTN-Hun, the RJ3 was located at nucleotide (nt) 9183, namely the 3' proximal end of the CP gene (nt. 8571-9371), thus leading to most (the first 613 nucleotides from the 5' proximal end) of the CP gene (801 bp) with a PVYN origin and PVYN-serotype; whereas in contrast, the RJ3 in PVYNTN-HN2 was located at nt 8572, consequently leading to a CP gene of PVYO origin and PVYO-serotype. The varied genome composition among PVYO, PVYN, PVYN:O, PVYNTN-HN1 and PVYNTN-HN2 made them useful for the investigation of possible roles of gene segment(s) in symptom formation on host plants. When Physalis floridana plants were infected with different PVY isolates, two types of symptoms were induced. PVYN and PVYNTN-HN1 induced mild symptoms (mainly mild mottling) whereas PVYO, PVYN:O and PVYNTN-HN2 induced serve symptoms including leaf and stem necrosis, leaf-drop and stunting. These results, together with a previous study using artificial PVY chimeras, demonstrate that the CP gene, especially the 5' proximal segment (nt 8572-9183), and/or CP likely determine the pathogenicity of PVY in P. floridana

Use of HRMA Proteins and Their Genes for Broad Range Protection of Plants Against Bacterial, Fungal and Viral Pathogens

The use of an avr gene hrmA to induce systematic acquired resistance in plant cells, plant seeds, plant tissues and plants is disclosed. Also disclosed is the use of low level expression of promoters in combination with the hrmA gene to provide broad-spectrum pathogen resistance in plant cells, plant seeds, plant tissues and plants