RNA-seq analysis reveals an early defense response to tomato leaf curl New Delhi virus in potato cultivar Kufri Bahar.

Potatoes in India are very susceptible to apical leaf curl disease, which causes severe symptoms and greater yield losses. Because the majority of potato cultivars are susceptible to the virus, it is crucial to discover sources of resistance and investigate the mechanism of resistance/susceptibility in potato cultivars. In this study, the gene expression profile of two potato cultivars, Kufri Bahar (resistant) and Kufri Pukhraj (susceptible), varying in their level of resistance to ToLCNDV, was analyzed using RNA-Seq. The Ion ProtonTM system was used to sequence eight RiboMinus RNA libraries from inoculated and uninoculated potato plants at 15 and 20 days after inoculation (DAI). The findings indicated that the majority of differentially expressed genes (DEGs) were cultivar-or time-specific. These DEGs included genes for proteins that interact with viruses, genes linked with the cell cycle, genes for proteins involved in defense, transcription and translation initiation factors, and plant hormone signaling pathway genes. Interestingly, defense responses were generated early in Kufri Bahar, at 15 DAI, which may have impeded the replication and spread of ToLCNDV. This research provides a genome-wide transcriptional analysis of two potato cultivars with variable levels of ToLCNDV resistance. At an early stage, we observed suppression of genes that interact with viral proteins, induction of genes associated with restriction of cell division, genes encoding defense proteins, AP2/ERF transcription factors, and altered expression of zinc finger protein genes, HSPs, JA, and SA pathway-related genes. Our findings add to a greater comprehension of the molecular basis of potato resistance to ToLCNDV and may aid in the development of more effective disease management techniques.

Spraying of dsRNA molecules derived from Phytophthora infestans, along with nanoclay carriers as a proof of concept for developing novel protection strategy for potato late blight.

BACKGROUND: Phytophthora infestans is a late blight-causing oomycetes pathogen. It rapidly evolves and adapts to the host background and new fungicide molecules within a few years of their release, most likely because of the predominance of transposable elements in its genome. Frequent applications of fungicides cause environmental concerns. Here, we developed target-specific RNA interference (RNAi)-based molecules, along with nanoclay carriers, that when sprayed on plants are capable of effectively reducing late blight infection. RESULTS: Targeted the genes unique to sporulation, early satge infection and the metabolism pathway stages based on in an our own microarray data. We used nanoclay as a carrier for sorbitol dehydrogenase, heat shock protein 90, translation elongation factor 1-α, phospholipase-D like 3 and glycosylphosphatidylinositol-anchored acidic serine-threonine-rich HAM34-like protein double-stranded (ds)RNAs, which were assessed by culture bioassay, detached leaf assay and spray methods, and revealed a reduction in growth, sporulation and symptom expression. Plants sprayed with multigene targeted dsRNA-nanoclay showed enhanced disease resistance (4% disease severity) and less sporulation (<1 × 103 ) compared with plants sprayed with dsRNA alone. CONCLUSION: The use of nanoclay with multigene targeted dsRNA was assumed to be involved in effective delivery, protection and boosting the action of RNAi as a spray-induced gene silencing approach (SIGS). A significant reduction in growth, sporulation, disease severity and decreased gene expression authenticates the effects of SIGS on late blight progression. This study demonstrated as a proof of concept the dsRNA-nanoclay SIGS approach, which could be used as an alternative to chemical fungicides and transgenic approaches to develop an environmentally friendly novel plant protection strategy for late blight. © 2022 Society of Chemical Industry.

An insight into the downstream analysis of RB gene in F1 RB potato lines imparting field resistance to late blight.

Earlier studies have shown that level of late blight resistance conferred by the classical R gene (RB Rpi-blb1) is dependent on genetic background of the recipient genotype. This was revealed in the analysis of late blight response that belonged to a group of F1 progeny obtained from the cross between Kufri Jyoti and SP951, which showed wide variation in late blight resistance response in spite of possessing the same RB gene. The global gene expression pattern in the RB potato lines was studied in response to late blight infection using cDNA microarray analysis to reveal the background effect. Leaf samples were collected at 0, 24, 72 and 120h post inoculation (hpi) with Phytophthora infestans for gene expression analysis using 61031 gene sequences. Significantly upregulated (1477) and downregulated (4245) genes common in the RB-transgenic F1 lines at 24 and 72 hpi were classified into several categories based on GO identifiers and majority of genes were assigned putative biological functions. Highest expression of an NBS-LRR along with protease, pectin esterase inhibitors, chaperones and reactive oxygen species genes were observed which affirmed a significant role of these categories in the defence response of RB-KJ lines. Results suggest that the immune priming of plant receptors are likely to be involved in stability and functionality of RB to induce resistance against P. infestans. This study is important for effective deployment of RB gene in the host background and contributes immensely to scientific understanding of R gene interaction with host protein complexes to regulate defence system in plants.

Genotypic background of the recipient plant is crucial for conferring RB gene mediated late blight resistance in potato.

BACKGROUND: Late blight, caused by oomycetes pathogen Phytophthora infestans (Mont.) de Bary, is the most devastating potato disease in the world. RB gene from Solanum bulbocastanum has been shown to impart broad spectrum resistance against P. infestans races. In this study Katahdin transgenic event SP951 was used as male parent to cross with the popular Indian potato cultivars viz., Kufri Bahar (KB) and Kufri Jyoti (KJ) to enhance the late blight resistance. RESULTS: Populations of 271 F1seedlings from the crosses KB × SP951 (87) and KJ × SP951 (184) were screened for inheritance of RB transgene through PCR and bioassay. Disease response based on AUDPC of different hybrid lines varied from immunity to complete susceptibility. High degree of resistance (<25% infection) was observed in KJ × SP951 derived seedlings (85.2%), whereas level of resistance in KB × SP951 (36.4% infection) derived seedlings was of low order. CONCLUSION: This study provides valuable genetic materials for development of potentially durable late blight resistant potato varieties. Besides, it also corroborates the fact that efficacy of R gene is not solely dependent on its presence in the variety but largely depends on the genetic background of the recipient genotype.

An insight into differentially regulated genes in resistant and susceptible genotypes of potato in response to tomato leaf curl New Delhi virus-[potato] infection.

Apical leaf curl disease, caused by tomato leaf curl New Delhi virus-[potato] (ToLCNDV-[potato]) is one of the most important viral diseases of potato in India. Genetic resistance source for ToLCNDV in potato is not identified so far. However, the cultivar Kufri Bahar is known to show lowest seed degeneration even under high vector levels. Hence, microarray analysis was performed to identify differentially regulated genes during ToLCNDV-[potato] infection in a resistant (Kufri Bahar) and a susceptible cultivar (Kufri Pukhraj). Under artificial inoculation conditions, in Kufri Pukhraj, symptom expressions started at 15days after inoculation (DAI) and then progressed to severe symptoms, whereas no or only very mild symptoms were observed in Kufri Bahar up to 35 DAI. Correspondingly, qPCR assay indicated a high viral load in Kufri Pukhraj and a very low viral load in Kufri Bahar. Microarray analysis showed that a total of 1111 genes and 2588 genes were differentially regulated (|log2 (Fold Change)|>2) in Kufri Bahar and Kufri Pukhraj, respectively, following ToLCNDV-[potato] infection. Gene ontology and mapman analyses revealed that these altered transcripts were involved in various biological & metabolic processes. Several genes with unknown functions were 5 to 100 fold expressed after virus infection and further experiments are necessary to ascertain their role in disease resistance or susceptibility. This study gives an insight into differentially regulated genes in response to ToLCNDV-[potato] infection in resistant and susceptible cultivars and could serve as the basis for the development of new strategies for disease management.

Expression profiling of potato cultivars with contrasting tuberization at elevated temperature using microarray analysis.

Temperature is one of the most significant factors affecting potato yield. Night temperature beyond 18-22 °C drastically reduces tuber formation, constraining potato cultivation in tropics and subtropics. Identification of genes and pathways affected by high temperature is crucial for developing thermo tolerant cultivars for these regions. In the present study, two cultivars with contrasting tuberization behavior at night temperatures (24 °C) were selected for gene expression analysis using a customized microarray chip representing 39,031 potato genes. A total of 2500 genes were differentially expressed on 21 d and 4096 genes on 14 d after stress. Gene ontology and pathway analysis provided insights into the probable biological processes and pathways governing tuberization at elevated temperature. Pathway maps were constructed to graphically represent the gene expression patterns. Genes associated with photosynthesis, hormonal activity, sugar transporters and transcription factors were differentially expressed. The results are presented and discussed in terms of tuberization at high temperature. The effect of high temperature on expression of genes controlling tuberization was also analyzed. This study provided useful information on potato tuberization at elevated temperature and make available a framework for further investigations into heat stress in potato.

Storage behaviour and cooking quality of Indian potato varieties.

Tubers of forty four indigenous potato varieties were assessed for storage behaviour at room temperature, tuber dry matter content and cooking quality during 2010, 2011 and 2012. The maximum, minimum temperatures and relative humidity during storage period ranged between 26 to 40 °C, 17-28 °C and 18 to 82 %, respectively. The lowest total weight loss was recorded in variety Kufri Pushkar (7.7 %) followed by Kufri Lalima (7.9 %), Kufri Surya (8.3 %), Kufri Red (9.2 %), Kufri Dewa, Kufri Sheetman (9.3 %), Kufri Chandramukhi, Kufri Jyoti (9.5 %), Kufri Sindhuri (9.6 %), Kufri Kuber (9.7 %), Kufri Chipsona-1 (9.8 %), Kufri Kundan (9.9 %) and Kufri Chamatkar (10.0 %). Highest tuber dry matter content (%) was observed in Variety Kufri Kundan (24.2) followed by Kufri Himsona (23.7), Kufri Frysona (23.6), Kufri Kuber (22.7), Kufri Chipsona-2 (22.3). Kufri Khasigaro (22.0), Kufri Sheetman (21.9), Kufri Chipsona-3 (21.7) and lowest in Variety Kufri Khyati and Kufri Pukhraj (16.1 %). Of the total varieties, 14 were adjudged as floury, 15 mealy, 14 waxy and one (Kufri Ashoka) as soggy. The total weight loss had highly significant and positive correlation with sprout weight/Kg tubers (r = 0.76**), physiological weight loss (r = 0.97**). Based on the results potato varieties namely, Kufri Chamatkar, Kufri Chipsona-1, Kufri Chandramukhi, Kufri Dewa, Kufri Jyoti, Kufri Kuber, Kufri Kundan, Kufri Lalima, Kufri Lauvkar, Kufri Pushkar, Kufri Red, Kufri Safed, Kufri Sheetman, Kufri Sindhuri possessed excellent keeping quality with medium to long tuber dormancy, low storage losses, medium to high tuber dry matter and good flavour. The information generated in this study can be utilized in the breeding programme. This can also help the farmer to choose and cultivate the potato varieties as per demand of the consumers.

Microarray analysis of gene expression patterns in the leaf during potato tuberization in the potato somatic hybrid Solanum tuberosum and Solanum etuberosum.

Genes involved in photoassimilate partitioning and changes in hormonal balance are important for potato tuberization. In the present study, we investigated gene expression patterns in the tuber-bearing potato somatic hybrid (E1-3) and control non-tuberous wild species Solanum etuberosum (Etb) by microarray. Plants were grown under controlled conditions and leaves were collected at eight tuber developmental stages for microarray analysis. A t-test analysis identified a total of 468 genes (94 up-regulated and 374 down-regulated) that were statistically significant (p ≤ 0.05) and differentially expressed in E1-3 and Etb. Gene Ontology (GO) characterization of the 468 genes revealed that 145 were annotated and 323 were of unknown function. Further, these 145 genes were grouped based on GO biological processes followed by molecular function and (or) PGSC description into 15 gene sets, namely (1) transport, (2) metabolic process, (3) biological process, (4) photosynthesis, (5) oxidation-reduction, (6) transcription, (7) translation, (8) binding, (9) protein phosphorylation, (10) protein folding, (11) ubiquitin-dependent protein catabolic process, (12) RNA processing, (13) negative regulation of protein, (14) methylation, and (15) mitosis. RT-PCR analysis of 10 selected highly significant genes (p ≤ 0.01) confirmed the microarray results. Overall, we show that candidate genes induced in leaves of E1-3 were implicated in tuberization processes such as transport, carbohydrate metabolism, phytohormones, and transcription/translation/binding functions. Hence, our results provide an insight into the candidate genes induced in leaf tissues during tuberization in E1-3.

A quantitative Real Time PCR based method for the detection of Phytophthora infestans causing Late blight of potato, in infested soil.

A fast and simple polymerase chain reaction method has been developed for detection of Phytophthora infestans oospores, the causal agent of Late blight of Potato in soil. The method involves the disruption of oospores by grinding dry soil, using abrasive properties, in the presence of glass powder and skimmed milk powder within a short time. The latter prevents loss of DNA by adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with the DNA. After phenol/chloroform extraction; the DNA is suitable for direct PCR amplification without a precipitation step. This amplification leads to detection of pathogen in infested soils before planting of crop. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. infestans detection to confirm positive inoculum level in potato seeds and elsewhere. With increasing amounts of standard DNA templates, the respective threshold cycle (Ct) values were determined and a linear relationship was established between these Ct values and the logarithm of initial template amounts. The method is rapid, cost efficient, and when combined with suitable internal controls can be applied to the detection and quantification of P. infestans oospores on a large-scale basis.

Analysis of plastome and chondriome genome types in potato somatic hybrids from Solanum tuberosum × Solanum etuberosum.

Cytoplasm types of the potato somatic hybrids from Solanum tuberosum × Solanum etuberosum were analysed using chloroplast (cp) and mitochondrial (mt) organelle genomes-specific markers. Of the 29 markers (15 cpDNA and 14 mtDNA) amplified in the 26 genotypes, 5 cpDNA (H3, NTCP4, NTCP8, NTCP9, and ALC1/ALC3) and 13 mtDNA markers showed polymorphism. The cluster analysis based on the mtDNA markers detected higher diversity compared with the cpDNA markers. Presence of new mtDNA fragments of the markers, namely, T11-2, Nsm1, pumD, Nsm3, and Nsm4, were observed, while monomorphic loci revealed highly conserved genomic regions in the somatic hybrids. The study revealed that the somatic hybrids had diverse cytoplasm types consisting predominantly of T-, W-, and C-, with a few A- and S-type cp genomes; and α-, ß-, and γ-type mt genomes. Somatic hybridization has unique potential to widen the cytoplasm types of the cultivated gene pools from wild species through introgression by breeding methods.

Analysis of genetic and epigenetic variation in in vitro propagated potato somatic hybrid by AFLP and MSAP marker

Background: Genetic and epigenetic changes (DNA methylation) were examined in the tissue-culture propagated interspecific potato somatic hybrids between dihaploid Solanum tuberosum and S. pinnatisectum. Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) were applied to detect the genetic and epigenetic changes, respectively in the somatic hybrids mother plants (1st cycle) and their regenerants (30th cycles sub-cultured). Results: To detect genetic changes, eight AFLP primer combinations yielded a total of 329 scorable bands of which 49 bands were polymorphic in both mother plants and regenerants. None of the scorable bands were observed in term of loss of original band of mother plant or gain of novel band in their regenerants. AFLP profiles and their cluster analysis based on the Jaccard’s similarity coefficient revealed 100% genetic similarity among the mother plant and their regenerants. On the other hand, to analyze epigenetic changes, eight MSAP primer pair combinations detected a few DNA methylation patterns in the mother plants (0 to 3.4%) and their regenerants (3.2 to 8.5%). Out of total 2320 MSAP sites in the mother plants, 2287 (98.6%) unmethylated, 21 (0.9%) fully methylated and 12 (0.5%) hemi-methylated, and out of total 2494 MSAP sites in their regenerants, 2357 (94.5%) unmethylated, 79 (3.1%) fully methylated and 58 (2.3%) hemi-methylated sites were amplified. Conclusion: The study concluded that no genetic variations were observed among the somatic hybrids mother plants and their regenerants by eight AFLP markers. However, minimum epigenetic variations among the samples were detected ranged from 0 to 3.4% (mother plants) and 3.2 to 8.5% (regenerants) during the tissue culture process.