Genome-wide identification and characterization of DIRIGENT gene family (CcDIR) in pigeonpea (Cajanus cajan L.) provide insights on their spatial expression pattern and relevance to stress response.

This study is a thorough characterization of pigeonpea dirigent gene (CcDIR) family, an important component of the lignin biosynthesis pathway. Genome-wide analysis identified 25 CcDIR genes followed by a range of analytical approaches employed to unravel their structural and functional characteristics. Structural examination revealed a classic single exon and no intron arrangement in CcDIRs contributing to our understanding on evolutionary dynamics. Phylogenetic analysis elucidated evolutionary relationships among CcDIR genes with six DIR sub-families, while motif distribution analysis displayed and highlighted ten conserved protein motifs in CcDIRs. Promoter analyses of all the dirigent genes detected 18 stress responsive cis-acting elements offering insights into transcriptional regulation. While spatial expression analyses across six plant tissues showed preferential expression of CcDIR genes, exposure to salt (CcDIR2 and CcDIR9) and herbivory (CcDIR1, CcDIR2, CcDIR3 and CcDIR11), demonstrated potential roles of specific DIRs in plant defense. Interestingly, increased gene expression during herbivory, also correlated with increased lignin content authenticating the specific response. Furthermore, exogenous application of stress hormones, SA and MeJA on leaves significantly induced the expression of CcDIRs that responded to herbivory. Taken together, these findings contribute to a comprehensive understanding of CcDIR genes impacting development and stress response in the important legume pigeonpea.

Cajanus Scarabaeoides Yellow Mosaic Virus, a New Bipartite Begomovirus Causing Yellow Mosaic Disease in Cajanus scarabaeoides in India.

Yellow mosaic disease of Cajanus scarabaeoides (L.) Thouars (CsYMD) was observed in up to 46% of C. scarabaeoides plants in the mungbean, urdbean, and pigeon pea fields from 22 districts of Chhattisgarh State, India, during 2017 to 2019. The symptoms were characterized by yellow mosaic on green leaves and yellow discoloration of leaves in advanced stages of the disease. Severely infected plants showed shortened internodal length and reduced leaf size. CsYMD was transmissible to healthy C. scarabaeoides and C. cajan by whitefly (Bemisia tabaci). The infected plants developed typical yellow mosaic symptoms on their leaves within 16 and 22 days of inoculation, respectively, suggesting a begomovirus etiology. Molecular analysis revealed that this begomovirus has a bipartite genome composed of DNA-A (2,729 nucleotides) and DNA-B (2,630 nucleotides). Sequence and phylogenetic analyses revealed that the nucleotide sequence of the DNA-A component had the highest identity of 81.1% with DNA-A of Rhynchosia yellow mosaic virus (RhYMV; NC_038885), followed by mungbean yellow mosaic virus (MN602427; 75.3%). DNA-B had the highest identity of 74.0% with DNA-B of RhYMV (NC_038886). As per ICTV guidelines, this isolate had <91% nucleotide identity with DNA-A of any of the begomoviruses reported; so, it is proposed as a new begomovirus species, tentatively named C. scarabaeoides yellow mosaic virus (CsYMV). After agroinoculation with DNA-A and DNA-B clones of CsYMV, all Nicotiana benthamiana plants developed leaf curl symptoms along with light yellowing symptoms 8 to 10 days after inoculation (DAI), while ∼60% of the C. scarabaeoides plants developed yellow mosaic symptoms similar to those observed in the field 18 DAI, thus fulfilling Koch's postulates. From these agro-infected C. scarabaeoides plants, CsYMV was transmissible to healthy C. scarabaeoides plants by B. tabaci. Apart from these hosts, CsYMV also infected and caused symptoms in mungbean and pigeon pea.

Cajanus platycarpus Flavonoid 3'5' Hydroxylase_2 (CpF3'5'H_2) Confers Resistance to Helicoverpa armigera by Modulating Total Polyphenols and Flavonoids in Transgenic Tobacco.

Pod borer Helicoverpa armigera, a polyphagus herbivorous pest, tremendously incurs crop damage in economically important crops. This necessitates the identification and utility of novel genes for the control of the herbivore. The present study deals with the characterization of a flavonoid 3'5' hydroxylase_2 (F3'5'H_2) from a pigeonpea wild relative Cajanus platycarpus, possessing a robust chemical resistance response to H. armigera. Though F3'5'H_2 displayed a dynamic expression pattern in both C. platycarpus (Cp) and the cultivated pigeonpea, Cajanus cajan (Cc) during continued herbivory, CpF3'5'H_2 showed a 4.6-fold increase vis a vis 3-fold in CcF3'5'H_2. Despite similar gene copy numbers in the two Cajanus spp., interesting genic and promoter sequence changes highlighted the stress responsiveness of CpF3'5'H_2. The relevance of CpF3'5'H_2 in H. armigera resistance was further validated in CpF3'5'H_2-overexpressed transgenic tobacco based on reduced leaf damage and increased larval mortality through an in vitro bioassay. As exciting maiden clues, CpF3'5'H_2 deterred herbivory in transgenic tobacco by increasing total flavonoids, polyphenols and reactive oxygen species (ROS) scavenging capacity. To the best of our knowledge, this is a maiden attempt ascertaining the role of F3'5'H_2 gene in the management of H. armigera. These interesting leads suggest the potential of this pivotal branch-point gene in biotic stress management programs.

The plant specialized metabolite epicatechin- 3-gallate (EC3G) perturbs lipid metabolism and attenuates fat accumulation in pigeonpea pod borer, Helicoverpa armigera.

Control of pod borer Helicoverpa armigera, a notorious polyphagous pest requires paramount attention with focus on environment-friendly management approaches. Overproduction of catechins (epigallocatechin-EGC and epicatechin-3-gallate-EC3G) in the pod borer-resistant pigeonpea wild relative, Cajanus platycarpus during continued herbivory prodded us to assess their underlying molecular effect on H. armigera. Significant reduction in larval and pupal growth parameters was observed when reared on artificial diet incorporated with 100 ppm EC3G vis a vis 100 ppm EGC and EGC + EC3G. Comparative RNAseq analyses of larvae that fed on normal and EC3G-incorporated diet revealed 62 differentially expressed genes dominated by detoxification and lipid metabolism. While lipase and fatty acid-binding protein 2-like were up-regulated, delta9-FADS-like involved in fatty acid synthesis was downregulated, indicating effect of EC3G on fat metabolism. Validation of RNAseq data by qPCR; midgut glutathione-S-transferase and esterase assays depicted increased lipolysis and reduced lipogenesis in EC3G-fed larvae. Additionally, differential accumulation of stearic acid and oleic acid in EC3G-fed and control larvae/adults ascertained perturbation in lipogenesis. Supported by modelling, molecular docking and simulations, we demonstrate the possible involvement of the insect adipokinetic hormone receptor (AKHR) in the EC3G-mediated response. The study demonstrates plant specialized metabolite EC3G as a potential candidate for H. armigera control.

Achieving maximum efficiency of Mungbean yellow mosaic India virus infection in mungbean by agroinoculation.

Mungbean is one of the important food legumes in the Indian-sub-continent. Yellow mosaic disease, caused by Mungbean yellow mosaic virus and Mungbean yellow mosaic India virus (MYMIV) poses a severe threat to its production. Agroinoculation has been the most preferred way to test the function of genomic components of these viruses. However, the available inoculation methods are not as efficient as whitefly transmission, thereby limiting their usage for screening and biological studies. We hereby report an efficient and reproducible agroinoculation method for achieving maximum (100%) efficiency using tandem repeat infectious agro-constructs of DNA A and DNA B of MYMIV. The present study targeted wounding of various meristematic tissues of root, shoot, parts of germinating seeds and also non-meristematic tissue of stem to test the suitable tissue types for maximum infection. Among the various tissues selected for, the inoculation on the epicotyl region showed maximum infectivity. Further, to enhance the infectivity of MYMIV, different concentrations of acetosyringone, incubation time and Agrobacterium cell density were also standardized. The incubation of wounded sprouted seeds in 1.0 OD of agroculture containing repeat construct of MYMIV for 2-4 h without acetosyringone followed by sowing in soil showed maximum infection of MYMIV within 10-12 days on the first trifoliate leaf. This standardized method is reproducible and has potential to screen germplasm lines and will be useful in mungbean biological/virological studies and breeding programmes. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03088-w.

Molecular analysis, infectivity and host range of Tomato leaf curl Karnataka virus associated with Corchorus yellow vein mosaic betasatellite.

Severe leaf curl disease of tomato (ToLCD) was noticed recently in the central parts of India and is an emerging threat to the cultivation of tomato. The genomic components of the begomovirus isolate, DNA A and betasatellite associated with ToLCD were cloned by rolling circle amplification method and sequenced. The sequence analysis revealed that the DNA A (2766 nt) of this isolate had the nucleotide identity of >91% with other strains of Tomato leaf curl Karnataka virus (ToLCKV), hence this isolate is proposed as a strain of ToLCKV, named as ToLCKV-Raipur. Similarly, the betasatellite molecule (1355 nt) had the highest identity of 91.1% with Corchorus yellow vein mosaic betasatellite (CoYVMB) and named as CoYVMB-Raipur. The full-length dimerized clones of these two genomic components were agroinoculated on natural (tomato), experimental (Nicotiana benthamiana) hosts and other 20 plant species belong to six different families. The severe leaf curl symptoms appeared only in the hosts, N. benthamiana, and in tomato inoculated with ToLCKV-Raipur alone and ToLCKV-Raipur with CoYVMB-Raipur after 8 and 16-18 days inoculation, respectively. This isolate was also transmissible to healthy tomato plants by whitefly from the tomato plant agroinoculated with ToLCKV-Raipur alone and with CoYVMB-Raipur and produced symptoms within 14-16 days after inoculation. Interestingly, this isolate infects horse gram and chilli by whitefly transmission and both the hosts showed positive for DNA A alone but not for betasatellite. Quantification of the genomic components of this isolate with the agroinoculated N. benthamiana samples by qRT-PCR results showed that the quantity of ToLCKV-Raipur was enhanced by three-fold while inoculated with CoYVMB-Raipur compared to ToLCKV-Raipur alone inoculated plants. However, CoYVMB-Raipur did not enhance the levels of ToLCKV-Raipur in the agroinoculated tomato plants. This is the first evidence of the natural co-occurrence of ToLCKV with betasatellite, CoYVMB causing ToLCD.

Distinct morpho-physiological and biochemical features of arid and hyper-arid ecotypes of Ziziphus nummularia under drought suggest its higher tolerance compared with semi-arid ecotype.

Tree species in the arid and semi-arid regions use various strategies to combat drought stress. Ziziphus nummularia (Burm. f.) Wight et Arn., native to the Thar Desert in India, is highly drought-tolerant. To identify the most drought-tolerant ecotype of Z. nummularia, one ecotype each from semi-arid (Godhra, annual rainfall >750 mm), arid (Bikaner, 250-350 mm) and hyper-arid (Jaisalmer, <150 mm) regions was selected along with two other Ziziphus species, Ziziphus mauritiana Lamk. and Ziziphus rotundifolia Lamk., and screened for parameters contributing to drought tolerance. Among these, Z. nummularia (Jaisalmer) (CIAHZN-J) was the most drought - tolerant. The tolerance nature of CIAHZN-J was associated with increased membrane stability, root length and number, length of hairs and thorns, root dry/fresh weight ratio, seed germination (at -0.5 MPa), proline content (31-fold), catalase and sugar content (two- to three-fold). Apart from these characteristics, it also exhibited the longest duration to reach highest cumulative drought stress rating, maintained higher relative water content for a longer period of time with reduced leaf size, leaf rolling and falling of older leaves, and displayed sustained shoot growth during drought stress. To determine drought tolerance in Ziziphus, we developed a morphological symptom-based screening technique in this study. Additionally, transcriptome profiling of CIAHZN-J in response to drought revealed the up-regulation of genes involved in sugar metabolism and transport, abscisic acid biosynthesis, osmoregulation, reactive oxygen species homeostasis and maintaining water potential. Expression profiles and semi-quantitative reverse transcription PCR results further correlated with the physiological and biochemical mechanisms. In conclusion, CIAHZN-J is an excellent genetic stock for the identification of drought-responsive genes and can also be deployed in crop improvement programs for drought tolerance.