Engineering of Potato for Tolerance to Biotic and Abiotic Stress,
The central dogma (CD) of molecular biology constitutes the transfer of genetic information from DNA to RNA to protein. Major CD processes governing genetic flow include the cell cycle, DNA replication, chromosome packaging, epigenetic changes, transcription, posttranscriptional alterations, translation, and posttranslational modifications. The CD processes are tightly regulated in plants to maintain their genetic integrity throughout the life cycle as well as to pass the genetic material to the next generation. Engineering of various CD processes involved in gene regulation will accelerate crop improvement to feed the growing world population. CRISPR technology enables programmable editing of CD processes to alter DNA, RNA, or protein, which would have been impossible in the past. Here, an overview of recent advancements in CRISPR tool development and CRISPR-based CD modulations that expedite basic and applied plant research is provided. Furthermore, CRISPR applications in major thriving areas, such as gene discovery (allele mining and cryptic gene activation), introgression (de novo domestication and haploid induction), and application of desired traits beneficial to farmers or consumers (biotic/abiotic stress-resilient crops, plant cell factories, and delayed senescence), are described. Finally, the global regulatory policies, challenges, and prospects for CRISPR-mediated crop improvement are summarized.
Northern blot analysis of Solanum tuberosum infected with potato leafroll luteovirus revealed the 6 kb genomic RNA and a major 2.3 kb subgenomic RNA. The 5' end of the subgenomic RNA was located at nucleotide 3653 in an intergenic region located at the centre of the viral genome upstream of three open reading frames (ORFs). Transient expression in tobacco and potato protoplasts of the beta-glucuronidase reporter gene fused to various putative regulatory sequences present in the subgenomic RNA was used to study their influence on expression levels. We observed a suppression of the amber stop codon separating the coat protein (CP) gene from a downstream ORF (56K protein), to a level of 0.9% to 1.3%. Translation initiation at the AUG of an ORF (17K protein) which is nested within the CP gene, exceeds translation of the CP gene itself by a factor of 7.
Transgenic potato plants expressing mutant alleles of PLRV ORF4, the gene for the movement protein pr17 of this luteovirus, were generated for broad-range protection against virus infection. When tested for protection against infection by PLRV, all transgenic lines showed a significant reduction of virus antigen. Potato lines accumulating N- or C-terminally extended PLRV pr17 mutant proteins were resistant to infection by the unrelated potato viruses PVY and PVX. Transgenic lines that did not express protein despite high transcript levels failed to exhibit virus resistance.
Mono- and polyclonal antibodies directed against different domains of the potato leafroll luteovirus (PLRV) P1 (ORF1) protein
were applied to the analysis of P1 expression during PLRV replication in planta. Western analyses detected P1 and a protein of ∼25 kDa (P1-C25) that accumulated to readily detectable amounts in PLRV-infected
plants, but was not detected by in vitro cell-free translation of P1. P1-C25 represents the C-terminus of P1 and is a proteolytic cleavage product produced during
P1 processing. On the basis of its molecular weight, the N-terminus of P1-C25 is either identical to or located adjacent to
the previously identified PLRV genome-linked protein, VPg. P1-C25 is not associated with virus particles, and subcellular
localization experiments detected P1-C25, but not P1, in the membrane and cytoplasmic fractions of PLRVinfected cells. In
addition, P1-C25 exhibits nucleic acid-binding properties. On the basis of its biosynthesis, localization and biochemical
properties, P1-C25 may facilitate the formation of P1/PLRV RNA complexes in which the spatial proximity allows for covalent
bond formation between PLRV RNA and VPg.
Transgenic tobacco (Nicotiana tabacum L.) plants expressing the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to investigate the influence of a localized change in mesophyll-bundle sheath plasmodesmal size exclusion limit on photosynthetic performance and on carbon metabolism and allocation. Under conditions of saturating irradiance, tobacco plants expressing the TMV-MP were found to have higher photosynthetic CO2-response curves compared with vector control plants. However, this difference was significant only in the presence of elevated CO2 levels. Photosynthetic measurements made in the green-house, under endogenous growth conditions, revealed that there was little difference between TMV-MP-expressing and control tobacco plants. However, analysis of carbon metabolites within source leaves where a TMV-MP-induced increase in plasmodesmal size exclusion limit had recently taken place established that the levels of sucrose, glucose, fructose and starch were considerably elevated above those present in equivalent control leaves. Although expression of the TMV-MP did not alter total plant biomass, it reduced carbon allocation to the lower region of the stem and roots. This difference in biomass distribution was clearly evident in the lower root-to-shoot ratios for the TMV-MP transgenic plants. Microinjection (dye-coupling) studies established that the TMV-MP-associated reduction in photosynthate delivery (allocation) to the roots was not due to a direct effect on root cortical plasmodesmata. Rather, this change appeared to result from an alteration in phloem transport from young source leaves in which the TMV-MP had yet to exert its influence over plasmodesmal size exclusion limits. These results are discussed in terms of the rate-limiting steps involved in sucrose movement into the phloem.
Plant productivity is greatly influenced by environmental stresses, such as freezing, drought, salinity and flooding. One of the ways in which tolerance to these factors can be achieved is by the transfer of genes encoding protective proteins or enzymes from other organisms. Key approaches currently being examined are engineered alterations in the amounts of osmolytes and osmoprotectants, saturation levels of membrane fatty acids, and rate of scavenging of reactive oxygen intermediates.
A broadly-applicable strategy is proposed for genetically engineering resistance to parasites. The strategy involves deriving resistance genes from the genome of the parasite itself. Key gene products from the parasite, if present in a dysfunctional form, in excess, or at the wrong developmental stage, should disrupt the function of the parasite while having minimal affect on the host. Therefore, resistance might be routinely achieved by cloning the appropriate parasite gene, modifying its expression if necessary, and transforming it into the host genome. The QB bacteriophage is used to illustrate, specifically, how parasite-derived resistance might be engineered. Examples are given of pathogen-derived resistance as it already functions in nature, and potential applications of this strategy in agriculture are discussed. The advantages and limitations of parasite-derived resistance are outlined.
Modification of the genetic content of cultured cells or of whole animals is now a key strategy in both basic biological research and applied biotechnology. Yet obtaining the desired level and specificity of expression of an introduced gene remains highly problematic. One solution could be to couple expression of a transgene to that of an appropriate intact genomic locus. The identification and functional characterization of RNA sequences known as internal ribosome entry sites now offer the possibility of achieving precise control of transgene expression through the generation of dicistronic fusion mRNAs.
The 17 kDa protein (pr17) of potato leafroll luteovirus is translated from a subgenomic PLRV RNA by internal translation initiation and binds to single-stranded nucleic acids (E. Tacke, D. Prüfer, J. Schmitz, and E. Rohde, 1991, J. Gen. Virol. 72, 2035-2038). Chemical crosslinking of in vitro expressed pr17 provided evidence for the preferential formation of pr17 homodimers which were also detected in PLRV-infected potato plants and isolated from potato lines expressing the PLRV pr17 transgene. Mutation analysis identified an amphipathic alpha-helix within the acidic amino-terminus of pr17 which acts as the domain for protein/protein interactions. Pr17 was predominantly associated with subcellular fractions enriched for nuclei, chloroplasts, mitochondria, and membranous structures. In addition it was shown that pr17 was phosphorylated in planta and that this modification did not inhibit binding of the protein to nucleic acids.
Publisher Summary Molecular analysis has shown that the genomes of luteoviruses combine most of the strategies used to express the monopartite genomes of (+) sense ssRNA viruses. Moreover, luteovirus genomes have clearly evolved by recombination among blocks of coding sequence derived from distinct ancestral viruses. Thus, despite the difficulties of studying these viruses, many interesting molecular biology features have been demonstrated and would repay more intensive study. The biological features of luteoviruses are also unusual in that, except in peculiar circumstances, luteoviruses are confined to the phloem of their hosts and during transmission luteovirus particles interact with surfaces in their insect vectors to cross several cell boundaries. Both features can be explained in a general way, but the molecular bases for the properties are as yet poorly understood and are thus excellent candidates for more penetrating molecular study. It seems clear that much more progress can be anticipated on several fronts in the near future and that in many cases the knowledge gained should convey lessons applicable in the wider field of RNA virus molecular biology.
The 17 kDa protein (pr17), the phloem-limited movement protein (MP) of potato leafroll luteovirus (PLRV), is associated with membranous structures and localized to plasmodesmata [Tacke et al. (1993) Virology 197, 274-282; Schmitz, J. (1995) Ph.D. Thesis, University of Cologne]. In planta the protein is predominantly present in its phosphorylated form, but it is rapidly dephosphorylated during isolation under native conditions. In an effort to examine the nature of the protein kinase(s) involved in the phosphorylation reaction, pr17 deletion mutants were expressed as fusion proteins in a bacterial expression vector system and tested for their ability to be phosphorylated by potato membrane preparations as well as by commercially available kinases. A fusion protein containing the nucleic acid-binding, basic, C-proximal domain (pr17C1) was identified to be phosphorylated by a Ca2+- and phospholipid-dependent, membrane-associated protein kinase. This protein kinase activity was inhibited by the addition of (19-36) protein kinase C (PKC) inhibitory peptide, known to be a highly specific inhibitor of mammalian PKC. Moreover, also the mammalian PKC from rat was able to phosphorylate pr17 in vitro. The results suggest that phosphorylation of pr17 takes place at membranous structures, possibly at the deltoid plasmodesmata connecting the sieve cell-companion cell complex of the phloem, by the activity of PKC-related, membrane-associated protein kinase activity.
The potato leafroll virus (PLRV) 17-kDa protein (pr17), the putative movement protein for this phloem-limited luteovirus, was localized on ultrathin sections of leaves from PLRV-infected and transgenic potato plants. The transgenic plants expressed the entire viral genome from a full-length cDNA copy (PLRVfl) or only the gene encoding pr17 (ORF4) under the control of the cauliflower mosaic virus 35S promoter. Virus-infected and PLRVfl-transgenic plants developed symptoms typical of virus infection, whereas pr17-transgenic plants did not display symptoms or ultrastructural alterations. Immunogold electron microscopy using an anti-pr17-serum detected pr17 in plasmodesmata, in virus-induced vesicles, in mitochondria, and in chloroplasts of phloem cells, in PLRV-infected as well as PLRVfl-transgenic plants. In addition, in transgenic plants, pr17 was expressed in mesophyll cells (which are not infected by PLRV under natural conditions) and localized to the same sites as in phloem cells, except in plasmodesmata. In contrast, in pr17-transgenic plants the protein was never observed on organelles, but was almost exclusively associated with plasmodesmata of all leaf cell types, indicating that the targeting of pr17 to plasmodesmata is an intrinsic property of the protein. These results support the role of pr17 in PLRV movement.
Elucidating the role of viral genes in transgenic plants revealed that the movement protein (MP) from tobacco mosaic virus is responsible for altered carbohydrate allocation in tobacco and potato plants. To study whether this is a general feature of viral MPs, the movement protein MP17 of potato leafroll virus (PLRV), a phloem-restricted luteovirus, was constitutively expressed in tobacco plants. Transgenic lines were strongly reduced in height and developed bleached and sometimes even necrotic areas on their source leaves. Levels of soluble sugars and starch were significantly increased in source leaves. Yet, in leaf laminae the hexose-phosphate content was unaltered and ATP reduced to only a small extent, indicating that these leaves were able to maintain homeostatic conditions by compartmentalization of soluble sugars, probably in the vacuole. On the contrary, midribs contained lower levels of soluble sugars, ATP, hexose-phosphates and UDP-glucose supporting the concept of limited uptake and catabolism of sucrose in the phloem. The accumulation of carbohydrates led to a decreased photosynthetic capacity and carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase (rubisco) probably owing to decreased expression of photosynthetic proteins. In parallel, levels of pathogenesis-related proteins were elevated which may be the reason for the obtained limited resistance against the unrelated potato virus Y (PVY)N in the transgenic tobacco plants. Ultrathin sections of affected leaves harvested from 2-week-old plants revealed plasmodesmal alterations in the phloem tissue while plasmodesmata between mesophyll cells were indistinguishable from wild-type. These data favour the phloem tissue to be the primary site of PLRV MP17 action in altering carbohydrate metabolism.
The genetic information of potato leafroll virus (PLRV), a typical member of the subgroup 2 luteoviruses, is contained in
a single-stranded (+) sense RNA of ∼5.9 kb. A single subgenomic RNA (sgRNA1) of ∼2.3 kb has been characterized as the mRNA
for the 3′ clustered viral open reading frames ORF3, ORF3/5 and ORF4. Here we demonstrate by Northern blot analyses of polysomal
RNAs from PLRV-infected Solanum tuberosum and Physalis floridana plants that, as with luteoviruses belonging to subgroup 1, in planta synthesis of a second 0.8 kb subgenomic RNA (sgRNA2) increases the complexity of subgroup 2 luteoviral genomes significantly.
PLRV-specific hybridization probes as well as primer extension experiments map sgRNA2 to the 3′-end of the PLRV RNA genome
(positions 5190–5987). Similarly, for the closely related cucurbit aphid-borne yellows virus (CABYV) a sgRNA2 of similar size
and position (positions 4888–5669) was identified. PLRV sgRNA2 may code for two viral proteins of 7.1 (ORF6) and 14 kDa (ORF7)
respectively, while the CABYV proteins are 8.7 (ORF6) and 8.3 kDa (ORF7) in size, with PLRV ORF7 displaying nucleic acid binding
activity. In vivo experiments by transient expression of chimeric GUS fusions in potato protoplasts demonstrated that sgRNA2 functions as a
bicistronic mRNA with high expression of ORF6 and low translational efficiency for synthesis of ORF7.