Cas13d-mediated multiplex RNA targeting confers a broad-spectrum resistance against RNA viruses in potato.

CRISPR-Cas systems endow the bacterial and archaeal species with adaptive immune mechanisms to fend off invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13d has been harnessed to confer the protection against RNA viruses in diverse eukaryotic species. However a vast number of different viruses can potentially infect the same host plant resulting in mixed infection, thus necessitating the generation of crops with broad-spectrum resistance to multiple viruses. Here we report the repurposing of CRISPR/Cas13d coupled with an endogenous tRNA-processing system (polycistronic tRNA-gRNA, PTG) to target the multiple potato RNA viruses. Expression of Cas13d and four different gRNAs were observed in transgenic potato lines expressing the Cas13d/PTG construct. We show that the Cas13d/PTG transgenic plants exhibit resistance to either PVY, PVS, PVX or PLRV alone or two/three viruses simultaneously by reducing viral accumulation in plant cells. In sum, our findings provide an efficient strategy for engineering crops that can simultaneously resist infection by multiple RNA viruses.

StHAB1, a negative regulatory factor in abscisic acid signaling, plays crucial roles in potato drought tolerance and shoot branching.

Abscisic acid (ABA) is critical in drought tolerance and plant growth. Group A protein type 2C phosphatases (PP2Cs) are negative regulators of ABA signaling and plant adaptation to stress. Knowledge about the functions of potato group A PP2Cs is limited. Here, we report that the potato group A PP2C StHAB1 is broadly expressed in potato plants and strongly induced by ABA and drought. Suppression of StHAB1 enhanced potato ABA sensitivity and drought tolerance, whereas overexpression of the dominant mutant StHAB1G276D compromised ABA sensitivity and drought tolerance. StHAB1 interacts with almost all ABA receptors and the Snf1-Related Kinase OST1. Suppressing StHAB1 and overexpressing StHAB1G276D alter potato growth morphology; notably, overexpression of StHAB1G276D causes excessive shoot branching. RNA-sequencing analyses identified that the auxin efflux carrier genes StPIN3, StPIN5, and StPIN8 were up-regulated in StHAB1G276D-overexpressing axillary buds. Correspondingly, the auxin concentration was reduced in StHAB1G276D-overexpressing axillary buds, consistent with the role of auxin in repressing lateral branch outgrowth. The expression of BRANCHED1s (StBRC1a and StBRC1b) was unchanged in StHAB1G276D-overexpressing axillary buds, suggesting that StHAB1G276D overexpression does not cause axillary bud outgrowth via regulation of BRC1 expression. Our findings demonstrate that StHAB1 is vital in potato drought tolerance and shoot branching.

Global Screening and Functional Identification of Major HSPs Involved in PVY Infection in Potato.

HSP40 (also known as DnaJ), HSP70, and HSP90 are major heat shock protein (HSP) families that play critical roles in plant growth and development and stress adaption. Recently, several members of the three HSP families were reported to be widely involved in the plant host-virus interactions. However, their global expression profiles and core members recruited by viruses are largely unknown. In this study, a total of 89 StDnaJs were identified from a genome-wide survey, and their classification, phylogenetic relationships, chromosomal locations, and gene duplication events were further analyzed. Together with 20 StHSP70s and 7 StHSP90s previously identified in the potato genome, the global expression patterns of the members in 3 HSP families were investigated in 2 potato cultivars during Potato virus Y (PVY) infection using RNA-seq data. Of them, 16 genes (including 8 StDnaJs, 6 StHSP70s, and 2 StHSP90s) were significantly up- or downregulated. Further analysis using qRT-PCR demonstrated that 7 of the 16 genes (StDnaJ06, StDnaJ17, StDnaJ21, StDnaJ63, StHSP70-6, StHSP70-19, and StHSP90.5) were remarkably upregulated in the potato cultivar 'Eshu 3' after PVY infection, implying their potential roles in the potato-PVY compatible interaction. Subsequent virus-induced gene silencing (VIGS) assays showed that silencing of the homologous genes of StDnaJ17, StDnaJ21, StHSP70-6, and StHSP90.5 in Nicotiana. benthamiana plants dramatically reduced the accumulation of PVY, which indicated the four genes may function as susceptibility factors in PVY infection. This study provides candidate genes for exploring the mechanism of potato-PVY compatible interaction and benefits breeding work aiming to produce new cultivars with the ability to grow healthily under PVY infection.

Extreme Resistance to Potato Virus A in Potato Cultivar Barbara is Independently Mediated by Ra and Rysto.

Potato virus A (PVA) and potato virus Y (PVY) are two members of genus Potyvirus infecting potato crops worldwide. Host resistance offers an economical and effective means for the control or management of these viruses. In this study, 20 potato clones were screened for their resistance against PVA and PVY by mechanical or graft inoculation assay, and were explored for the relationship between extreme resistance genes Ra and Ry by the detection of molecular markers linked to Ryadg, Rysto, and Rychc. Six clones, including Barbara, Jizhangshu 8, Longshu 7, Longshu 8, M6, and Solara, were found to be extremely resistant to both PVA and PVY; three clones (AC142, Eshu 3, and Shepody) were deemed to be extremely resistant to PVA but susceptible to PVY. To further reveal the inheritance of the extreme resistance (ER) against PVA, a tetraploid F1 population of Barbara × F58050 (susceptible to both PVY and PVA) and a tetraploid BC1 population of BF145 (a PVA-resistant but PVY-susceptible progeny of Barbara × F58050) × F58050 were obtained. Phenotyping of the F1 and BC1 populations by graft inoculation with PVA showed segregation ratios of 3:1 and 1:1 (resistant:susceptible), respectively. These results suggest that two independent loci control ER against PVA in Barbara: one confers ER to both PVA and PVY and the other confers ER to PVA only. The deduced genotype of Barbara is RyryryryRararara.

Comparative transcriptome reveals distinct starch-sugar interconversion patterns in potato genotypes contrasting for cold-induced sweetening capacity.

Potato accumulates large amounts of soluble sugar during cold storage periods. However, a system based understanding of this process is still largely unknown. Here, we compared the dynamic cold-responded transcriptome of genotypes between cold-induced sweetening resistant (CIS-R) and cold-induced sweetening sensitive (CIS-S) in tubers. Comparative transcriptome revealed that activating the pathways of starch degradation, sucrose synthesis and hydrolysis could be common strategies in response to cold in both genotypes. Moreover, the variation in sugar accumulation between genotypes may be due to genetic differences in cold response, which could be mainly explained: CIS-R genotype was active in starch synthesis and attenuated in sucrose hydrolysis by promoting the coordinate expression of aseries ofgenes involved in starch-sugar interconversion. Additionally, transcription factors, the candidate master regulators of starch-sugar interconversion, were discussed. Taken together, this work has provided an avenue for studying the mechanism involved in the regulation of the CIS resistance.

Effect of excessive nitrogen on levels of amino acids and sugars, and differential response to post-harvest cold storage in potato (Solanum tuberosum L.) tubers.

Nitrogen (N) is an important nutrient for increased potato tuber yield. However, excessive N can decrease tuber quality. Furthermore, the impact of optimal and higher N levels of potato tuber metabolic profile at harvest and cold storage remains unclear. This study aimed to investigate the metabolic profiling of free amino acids and sugars in potato tubers affected by different nitrogen levels (optimal, ON; and excessive, EN) at harvest (AH) and cold storage (CS) (~4 °C, 4 weeks) through untargeted GC-TOF-MS, and targeted UHPLC-QqQ-MS. Carbohydrate content and vacuolar invertase activity (IV) were determined. Principal component analysis of metabolite data indicated a distinct separation between ON and EN treatments at harvest and cold storage. Multivariate data analysis revealed that sucrose, reducing sugars, and free asparagine were the most altered metabolites (VIP > 1 and P < 0.05), which were involved in starch and sucrose metabolism, and alanine, aspartate and glutamate metabolism. At harvest, the absolute contents of various free amino acids including asparagine were higher (by 1.3-1.5 fold) in the EN treatment than ON treatment, and this difference was maintained at 4-week cold storage. Under the EN treatment, tuber maturity was reduced, and sucrose accumulation was increased at harvest, while IV was increased after cold storage, reducing sugar also accumulated. These results highlighted the negative effects of EN on free amino acid and sugars metabolism in the post-harvest tubers and provided useful information for understanding the underpinning physiological mechanisms.

Rapid and sensitive detection of potato virus Y by isothermal reverse transcription-recombinase polymerase amplification assay in potato.

In this study, an isothermal reverse transcription-recombinase polymerase amplification (RT-RPA) assay was developed for the efficient and accurate detection of potato virus Y (PVY) under isothermal conditions. This RT-RPA assay was more efficient than the conventional reverse transcription-polymerase chain reaction (RT-PCR) assay as the amplification reaction can be completed in less than 20 min. Moreover, unlike PCR that requires a thermocycler to carry out the DNA amplification through specific temperature phases, RPA assay could be performed under an isothermal condition at a temperature ranging from 25 to 40 °C. A simple instrumentation such as a heating block or a water bath or even anon-instrumental condition such as human hands or a benchtop inside/outside a room during the summer could satisfy the temperature requirement of RPA. The sensitivity of this assay was equivalent to that of the conventional RT-PCR, and the virus can be detected in a minimum of 2 pg of total RNA extracted from the PVY infected potato leaf tissues. The efficacy of the newly developed RT-RPA was then evaluated using field potato leaf and dormancy-broken sprout samples upon enzyme-linked immunosorbent assay (ELISA) screening. Of the 164 PVY-ELISA-positive samples, RT-RPA detected 157 whereas simplex RT-PCR detected 160 and multiplex RT-PCR detected 154. Of the 74 randomly selected PVY-ELISA-negative samples, RT-RPA, simplex RT-PCR and multiplex RT-PCR led to 1, 1 and 0 positive detections, receptively. Overall, RT-RPA and the two RT-PCR assays as well as ELISA exhibited an agreement of 96.6-98.7%, thus demonstrating the suitability of RT-RPA for large scale detection of PVY, irrespective of the strain type of the virus.

Generation of virus-resistant potato plants by RNA genome targeting.

CRISPR/Cas systems provide bacteria and archaea with molecular immunity against invading phages and foreign plasmids. The class 2 type VI CRISPR/Cas effector Cas13a is an RNA-targeting CRISPR effector that provides protection against RNA phages. Here we report the repurposing of CRISPR/Cas13a to protect potato plants from a eukaryotic virus, Potato virus Y (PVY). Transgenic potato lines expressing Cas13a/sgRNA (small guide RNA) constructs showed suppressed PVY accumulation and disease symptoms. The levels of viral resistance correlated with the expression levels of the Cas13a/sgRNA construct in the plants. Our data further demonstrate that appropriately designed sgRNAs can specifically interfere with multiple PVY strains, while having no effect on unrelated viruses such as PVA or Potato virus S. Our findings provide a novel and highly efficient strategy for engineering crops with resistances to viral diseases.

High Resolution DNA Melting Assays for Detection of Rx1 and Rx2 for High-Throughput Marker-Assisted Selection for Extreme Resistance to Potato virus X in Tetraploid Potato.

Assessment of the existing PCR-gel electrophoresis-based methods for detection of Rx1 and Rx2, the genes that independently control extreme resistance (ER) to Potato virus X (PVX), indicated that the 5Rx1F/5Rx1R primer pair led to reliable detection of Rx1, whereas the 106Rx2F/106Rx2R primer pair detected Rx2 despite some nonspecific reactions in potato clones/cultivars without Rx2. However, the methodology is time consuming and does not differentiate the absence of Rx1/Rx2 from a failed PCR reaction. A newly designed primer pair that targets Rx1 and Rx2 as well as rx1 and rx2 produced an amplicon for all alleles. When the primer pair was combined with 5Rx1F/5Rx1R, respective amplicons were produced, although they were not distinguishable by regular agarose gel electrophoresis. When subjected to a high-resolution DNA melting (HRM) assay, two distinct melting profiles for Rx1 and rx1, respectively, were detected. Triplex PCR-gel electrophoresis and -HRM assay for detection of Rx1, Rx2, and rx1/rx2 were also performed. The efficacy of the HRM assays were validated in potato cultivars/clones with known phenotypes, indicating its potential for high-throughput selection of potato clones/cultivars carrying Rx1 or Rx2. Duplex PCR-HRM assays of over 600 progeny from 12 crosses involving various parents correctly detected the presence or absence of Rx1 in each progeny, allowing accurate prediction of the phenotype. Progeny that tested positive for Rx1 by HRM exhibited ER to PVX whereas progeny that tested negative for Rx1 were susceptible to PVX infection. The genotype of each parent and the possible presence of Nx in two Rx1-possessing parents are also discussed.