Long non-coding RNA-mediated epigenetic response for abiotic stress tolerance in plants.

Plants perceive environmental fluctuations as stress and confront several stresses throughout their life cycle individually or in combination. Plants have evolved their sensing and signaling mechanisms to perceive and respond to a variety of stresses. Epigenetic regulation plays a critical role in the regulation of genes, spatiotemporal expression of genes under stress conditions and imparts a stress memory to encounter future stress responses. It is quintessential to integrate our understanding of genetics and epigenetics to maintain plant fitness, achieve desired genetic gains with no trade-offs, and durable long-term stress tolerance. The long non-coding RNA >200 nts having no coding potential (or very low) play several roles in epigenetic memory, contributing to the regulation of gene expression and the maintenance of cellular identity which include chromatin remodeling, imprinting (dosage compensation), stable silencing, facilitating nuclear organization, regulation of enhancer-promoter interactions, response to environmental signals and epigenetic switching. The lncRNAs are involved in a myriad of stress responses by activation or repression of target genes and hence are potential candidates for deploying in climate-resilient breeding programs. This review puts forward the significant roles of long non-coding RNA as an epigenetic response during abiotic stresses in plants and the prospects of deploying lncRNAs for designing climate-resilient plants.

Identification and Functional Characterization of Two Major Loci Associated with Resistance against Brown Planthoppers (Nilaparvata lugens (Stål)) Derived from Oryza nivara.

The brown planthopper (BPH) is a highly destructive pest of rice, causing significant economic losses in various regions of South and Southeast Asia. Researchers have made promising strides in developing resistance against BPH in rice. Introgression line RPBio4918-230S, derived from Oryza nivara, has shown consistent resistance to BPH at both the seedling and adult stages of rice plants. Segregation analysis has revealed that this resistance is governed by two recessive loci, known as bph39(t) and bph40(t), contributing to 21% and 22% of the phenotypic variance, respectively. We later mapped the genes using a backcross population derived from a cross between Swarna and RPBio4918-230S. We identified specific marker loci, namely RM8213, RM5953, and R4M17, on chromosome 4, flanking the bph39(t) and bph40(t) loci. Furthermore, quantitative expression analysis of candidate genes situated between the RM8213 and R4M17 markers was conducted. It was observed that eight genes exhibited up-regulation in RPBio4918-230S and down-regulation in Swarna after BPH infestation. One gene of particular interest, a serine/threonine-protein kinase receptor (STPKR), showed significant up-regulation in RPBio4918-230S. In-depth sequencing of the susceptible and resistant alleles of STPKR from Swarna and RPBio4918-230S, respectively, revealed numerous single nucleotide polymorphisms (SNPs) and insertion-deletion (InDel) mutations, both in the coding and regulatory regions of the gene. Notably, six of these mutations resulted in amino acid substitutions in the coding region of STPKR (R5K, I38L, S120N, T319A, T320S, and F348S) when compared to Swarna and the reference sequence of Nipponbare. Further validation of these mutations in a set of highly resistant and susceptible backcross inbred lines confirmed the candidacy of the STPKR gene with respect to BPH resistance controlled by bph39(t) and bph40(t). Functional markers specific for STPKR have been developed and validated and can be used for accelerated transfer of the resistant locus to elite rice cultivars.

First Report of the Association of Zygocactus virus X with Dragon Fruit (Hylocereus spp.) plants from Telangana, India.

Dragon fruit (Hylocereus spp.) a member of the family Cactaceae, is widely cultivated throughout the world, includingspan style="font-family:'Times New Roman'; letter-spacing:0.05pt; color:#333333"> India. During 2020-2021 crop growing season, mosaic symptoms were observed on the cladodes of dragon fruit plants (Purple Pink cultivar: 1-2% disease incidence) grown at a farmer's field of Telangana, India (Fig. S1 a). The symptomatic cladodes (n= 4), observed under leaf-dip electron microscopy (Zuchmaan and Zellnig, 2009) at Indian Agricultural Research Institute, New Delhi, revealed the presence of flexuous rod- shaped virus-like particles (Fig S1 b). Virus particles were of 580 x 13 nm size, corresponding to the genus Potexvirus. For further confirmation, the total RNA isolated from symptomatic cladodes using a NucleoSpin RNA Plant Mini kit (Macherey-Nagel). Subsequently, a reverse transcription polymerase chain reaction (RT-PCR) was performed using the PrimeScript 1st strand cDNA Synthesis Kit (Takara Bio). The cDNA was further amplified with the primers specific to coat protein (CP) gene of four different species of the genus Potexvirus known to infect members of Cactaceae family. Four sets of primers were used for detection, viz., Cactus virus X (CVX) (F, 5'-ATGTCTACTACTGGAGTCCA-3'; R, 5'-CTACTCAGGGCCTGGGAGAA-3'); Pitaya virus X (PiVX) (F, 5'-ATGGCTACTCAAACAGCACAA-3'; R, 5'-CTACTCTGGGGAGGGAAG-3'); Schlumbergera virus X (SchVX) (F, 5'-ATGTCGACCACTCCATCTTC-3'; R, 5'-TTATTCAGGGGATGGTAGTA-3') and Zygocactus virus X (ZyVX) (F, 5'-ATGTCTAACACTGCAGGAGT-3'; R, TCATTC GGGACCCGGTAGGA-3') (Duarte et al., 2008; Janssen et al., 2021; Parameswari et al., 2021), by following the PCR profile (Park et al., 2018). The species-specific primers of CVX, PiVX and SchVX did not amplify any amplicon, whereas the primers specific to ZyVX at nucleotide position 5841-6521 from complete CP gene have resulted in amplification of expected size (~680 base pairs) from all the samples. The gel-purified RT-PCR products were cloned into a pDrive cloning vector (Qiagen, Germany) and sequenced bi-directionally using Sanger sequencing. The resultant sequences (681 nt) of the CP gene showed 98% (nucleotide) and 100% (amino acid) sequence similarity with the CP gene sequence (Accession No: KY581590) of ZyVX. Hence, one representative sequence was deposited to the NCBI GenBank database as ZyVX-DPC isolate (Accession number- OK415019). The Neighbour Joining Phylogenetic Tree constructed using MEGA6 software (Tamura et al. 2013) showed grouping of Indian ZyVX-DPC isolate with the previously reported ZyVX isolates from Korea, Taiwan, China and Germany (Fig. S1c). These results confirmed the association of ZyVX with the symptomatic cladodes of dragon fruit plants collected from Telangana, India. Earlier studies revealed that ZyVX is a member of the genus Potexvirus known to infect dragon fruit plants from Brazil and China (Duarte et al., 2008). In India until now, anthracnose disease (Colletotrichum siamense) and CVX from Hylocereus spp. were reported (Abirami et al., 2019; Parameswari et al., 2021). To the best of our knowledge, this is the first report of ZyVX infection on dragon fruit in India. The draon fruit, being vegetatively propagated and with increasing cultivable area in India (Abirami et al, 2019), the present study gains significance. Further studies on mode of virus transmission, estimation of crop yield losses, host range studies and finding out source of resistance are essential.

Coordinated Action of RTBV and RTSV Proteins Suppress Host RNA Silencing Machinery.

RNA silencing is as an adaptive immune response in plants that limits the accumulation or spread of invading viruses. Successful virus infection entails countering the RNA silencing machinery for efficient replication and systemic spread in the host. The viruses encode proteins with the ability to suppress or block the host silencing mechanism, resulting in severe pathogenic symptoms and diseases. Tungro is a viral disease caused by a complex of two viruses and it provides an excellent system to understand the host and virus interactions during infection. It is known that Rice tungro bacilliform virus (RTBV) is the major determinant of the disease while Rice tungro spherical virus (RTSV) accentuates the symptoms. This study brings to focus the important role of RTBV ORF-IV in disease manifestation, by acting as both the victim and silencer of the RNA silencing pathway. The ORF-IV is a weak suppressor of the S-PTGS or stable silencing, but its suppression activity is augmented in the presence of specific RTSV proteins. Among these, RTBV ORF-IV and RTSV CP3 proteins interact with each other. This interaction may lead to the suppression of localized silencing as well as the spread of silencing in the host plants. The findings present a probable mechanistic glimpse of the requirement of the two viruses in enhancing tungro disease.

Increased Catalase Activity and Maintenance of Photosystem II Distinguishes High-Yield Mutants From Low-Yield Mutants of Rice var. Nagina22 Under Low-Phosphorus Stress.

An upland rice variety, Nagina22 (N22) and its 137 ethyl methanesulfonate (EMS)-induced mutants, along with a sensitive variety, Jaya, was screened both in low phosphorus (P) field (Olsen P 1.8) and in normal field (Olsen P 24) during dry season. Based on the grain yield (YLD) of plants in normal field and plants in low P field, 27 gain of function (high-YLD represented as hy) and 9 loss of function (low-YLD represented as ly) mutants were selected and compared with N22 for physiological and genotyping studies. In low P field, hy mutants showed higher P concentration in roots, leaves, grains, and in the whole plant than in ly mutants at harvest. In low P conditions, F v/F m and qN were 24% higher in hy mutants than in ly mutants. In comparison with ly mutants, the superoxide dismutase (SOD) activity in the roots and leaves of hy mutants in low P fields was 9% and 41% higher at the vegetative stage, respectively, but 51% and 14% lower in the roots and leaves at the reproductive stage, respectively. However, in comparison with ly mutants, the catalase (CAT) activity in the roots and leaves of hy mutants in low P fields was 35% higher at the vegetative stage and 15% and 17% higher at the reproductive stage, respectively. Similarly, hy mutants in low P field showed 20% and 80% higher peroxidase (POD) activity in the roots and leaves at the vegetative stage, respectively, but showed 14% and 16% lower POD activity at the reproductive stage in the roots and leaves, respectively. Marker trait association analysis using 48 simple sequence repeat (SSR) markers and 10 Pup1 gene markers showed that RM3648 and RM451 in chromosome 4 were significantly associated with grain YLD, tiller number (TN), SOD, and POD activities in both the roots and leaves in low P conditions only. Similarly, RM3334 and RM6300 in chromosome 5 were associated with CAT activity in leaves in low P conditions. Notably, grain YLD was positively and significantly correlated with CAT activity in the roots and shoots, F v/F m and qN in low P conditions, and the shoots' P concentration and qN in normal conditions. Furthermore, CAT activity in shoots was positively and significantly correlated with TN in both low P and normal conditions. Thus, chromosomal regions and physiological traits that have a role in imparting tolerance to low P in the field were identified.

Genotype × Environment interactions of Nagina22 rice mutants for yield traits under low phosphorus, water limited and normal irrigated conditions.

Multi environment testing helps identify stable genotypes especially for adverse abiotic stress situations. In the era of climate change and multiple abiotic stresses, it becomes important to analyze stability of rice lines under both irrigated and stress conditions. Mutants are an important genetic resource which can help in revealing the basis of natural variation. We analyzed 300 EMS induced mutants of aus rice cultivar Nagina22 (N22) for their G × E interaction and stability under low phosphorus (P), water limited and irrigated conditions. Environmental effect and interaction were more significant than genotypic contribution on grain yield (GY), productive tillers (TN) and plant height (PH) under these three environmental conditions in dry season, 2010. GY and TN were more affected by low P stress than by water limited condition, but PH was not significantly different under these two stresses. Mutants G17, G209, G29, G91, G63 and G32 were stable for GY in decreasing order of stability across the three environments but G254 and G50 were stable only in low P, G17 and G45 only in water limited and G295 and G289 only in normal irrigated condition. We then selected and evaluated 3 high yielding mutants, 3 low yielding mutants and N22 for their stability and adaptability to these 3 environments in both wet and dry seasons for six years (2010-2015). The most stable lines based on the combined analysis of 12 seasons were G125 (NH210) under normal condition, G17 (NH686), G176 (NH363) and G284 (NH162) in low P condition and G176 (NH363) under water limited condition. G176 was the best considering all 3 conditions. When screened for 15 Pup1 gene-specific markers, G176 showed alleles similar to N22. While two other low-P tolerant lines G17 and G65 showed N22 similar alleles only at k-1 and k-5 but a different allele or null allele at 13 other loci. These stable mutants are a valuable resource for varietal development and to discover genes for tolerance to multiple abiotic stresses.

Expression patterns of QTL based and other candidate genes in Madhukar × Swarna RILs with contrasting levels of iron and zinc in unpolished rice grains.

BACKGROUND: Identifying QTLs/genes for iron and zinc in rice grains can help in biofortification programs. Genome wide mapping showed 14 QTLs for iron and zinc concentration in unpolished rice grains of F7 RILs derived from Madhukar × Swarna. One line (HL) with high Fe and Zn and one line (LL) with low Fe and Zn in unpolished rice were compared with each other for gene expression using qPCR. 7 day old seedlings were grown in Fe+ and Fe- medium for 10 days and RNA extracted from roots and shoots to determine the response of 15 genes in Fe- conditions. RESULTS: HL showed higher upregulation than LL in shoots but LL showed higher upregulation than HL in roots. YSL2 was upregulated only in HL roots and YSL15 only in HL shoots and both up to 60 fold under Fe- condition. IRT2 and DMAS1 were upregulated 100 fold and NAS2 1000 fold in HL shoot. NAS2, IRT1, IRT2 and DMAS1 were upregulated 40 to 100 fold in LL roots. OsZIP8, OsNAS3, OsYSL1 and OsNRAMP1 which underlie major Fe QTL showed clear allelic differences between HL and LL for markers flanking QTL. The presence of iron increasing QTL allele in HL was clearly correlated with high expression of the underlying gene. OsZIP8 and OsNAS3 which were within major QTL with increasing effect from Madhukar were 8 fold and 4 fold more expressed in HL shoot than in LL shoot. OsNAS1, OsNAS2, OsNAS3, OsYSL2 and OsYSL15 showed 1.5 to 2.5 fold upregulation in flag leaf of HL when compared with flag leaf of Swarna. CONCLUSION: HL and LL differed in root length, Fe concentration and expression of several genes under Fe deficiency. The major distinguishing genes were NAS2, IRT2, DMAS1, and YSL15 in shoot and NAS2, IRT1, IRT2, YSL2, and ZIP8 in roots. The presence of iron increasing QTL allele in HL at marker locus close to genes also increased upregulation in HL.