Genome-wide identification and expression analysis of two component system genes in Cicer arietinum.

The two-component system (TCS) plays an important role in signal transduction pathways, cytokinin signaling and stress resistance of prokaryotes and eukaryotes. It is comprised of three types of proteins in plants; histidine kinases (HKs), histidine phosphotransfer proteins (HPs) and response regulators (RRs). Chickpea (Cicer arietinum L.) is one of the most important legume crops worldwide with special economic value in semi-arid tropics. Availability of complete genome sequence of chickpea presents a valuable resource for comparative analysis among angiosperms. In current study, Arabidopsis thaliana and Oryza sativa were used as reference plant species for comparative genomics analysis with C. arietinum. A genome-wide computational survey enabled us to identify putative members of TCS protein family including 18HKs, 26 RRs (7 type-A, 7 type-B, 2 type C and 10 pseudo) and 7 HPs (5 true and 2pseudo) genes in chickpea. The predicted TCS genes displayed family specific intron/exon organization and were randomly distributed across all the eight chromosomes. Comparative phylogenetic and evolutionary analysis suggested a variable conservation of TCS genes in relation to mono/dicot model plants and segmental duplication was the principal route of expansion for this family in chickpea. The promoter regions of TCS genes exhibited several abiotic stress-related cis-elements indicating their involvement in abiotic stress response. The expression analysis of TCS genes demonstrated stress (drought, heat, osmotic and salt) specific differential expression. Current study provides insight into TCS genes in C. arietinum, which will be helpful for further functional analysis of these genes in response to different abiotic stresses.

The Arabidopsis GPI-Anchored LTPg5 Encoded by At3g22600 Has a Role in Resistance against a Diverse Range of Pathogens.

Arabidopsis contains 34 genes for glycosylphosphatidylinositol (GPI)-anchored LTPg proteins. A motif analysis has placed these into four groups. With one exception, all are produced with a signal peptide and are most likely attached to the cell membrane via the GPI anchor. Several of the LTPg genes across the four groups are downregulated in syncytia induced by the beet cyst nematode Heterodera schachtii. We have here studied At3g22600 encoding LTPg5, which is the most strongly downregulated LTPg gene. It is mainly expressed in roots, and a promoter::GUS line was used to confirm the downregulation in syncytia and also showed downregulation in galls of the root knot nematode Meloidogyne incognita. In contrast, infection with bacteria (Pseudomonas syringae) and fungi (Botrytis cinerea) led to the induction of the gene in leaves. This diverse regulation of LTPg5 indicated a role in resistance, which we confirmed with overexpression lines and a T-DNA mutant. The overexpression lines were more resistant to both nematode species and to P. syringae and B. cinerea, while a knock-out mutant was more susceptible to H. schachtii and P. syringae. Thus, LTPg5 encoded by At3g22600 is part of the Arabidopsis resistance mechanism against pathogens. LTPg5 has probably no direct antimicrobial activity but could perhaps act by associating with a receptor-like kinase, leading to the induction of defense genes such as PR1.

The Arabidopsis RboHB Encoded by At1g09090 Is Important for Resistance against Nematodes.

Reactive oxygen species are a byproduct of aerobic metabolic processes but are also produced by plants in defense against pathogens. In addition, they can function as signaling molecules that control various aspects of plant life, ranging from developmental processes to responses to abiotic and biotic stimuli. In plants, reactive oxygen species can be produced by respiratory burst oxidase homologues. Arabidopsis contains 10 genes for respiratory burst oxidase homologues that are involved in different aspects of plant life. Plant pathogenic cyst nematodes such as Heterodera schachtii induce a syncytium in the roots of host plants that becomes a feeding site which supplies nutrients throughout the life of the nematode. In line with this function, the transcriptome of the syncytium shows drastic changes. One of the genes that is most strongly downregulated in syncytia codes for respiratory burst oxidase homologue B. This gene is root-specific and we confirm here the downregulation in nematode feeding sites with a promoter::GUS (ß-glucuronidase) line. Overexpression of this gene resulted in enhanced resistance against nematodes but also against leaf-infecting pathogens. Thus, respiratory burst oxidase homologue B has a role in resistance. The function of this gene is in contrast to respiratory burst oxidase homologues D and F, which have been found to be needed for full susceptibility of Arabidopsis to H. schachtii. However, our bioinformatic analysis did not find differences between these proteins that could account for the opposed function in the interaction with nematodes.

Overexpression of the transcription factor RAP2.6 leads to enhanced callose deposition in syncytia and enhanced resistance against the beet cyst nematode Heterodera schachtii in Arabidopsis roots.

BACKGROUND: Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is their source of nutrients throughout their life. A transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that gene expression in the syncytium is different from that of the root with thousands of genes upregulated or downregulated. Among the downregulated genes are many which code for defense-related proteins. One gene which is strongly downregulated codes for the ethylene response transcription factor RAP2.6. The genome of Arabidopsis contains 122 ERF transcription factor genes which are involved in a variety of developmental and stress responses. RESULTS: Expression of RAP2.6 was studied with RT-PCR and a promoter::GUS line. During normal growth conditions the gene was expressed especially in roots and stems. It was inducible by Pseudomonas syringae but downregulated in syncytia from a very early time point on. Overexpression of the gene enhanced the resistance against H. schachtii which was seen by a lower number of nematodes developing on these plants as well as smaller syncytia and smaller female nematodes. A T-DNA mutant had a reduced RAP2.6 transcript level but this did not further increase the susceptibility against H. schachtii. Neither overexpression lines nor mutants had an effect on P. syringae. Overexpression of RAP2.6 led to an elevated expression of JA-responsive genes during early time points after infection by H. schachtii. Syncytia developing on overexpression lines showed enhanced deposition of callose. CONCLUSIONS: Our results showed that H. schachtii infection is accompanied by a downregulation of RAP2.6. It seems likely that the nematodes use effectors to actively downregulate the expression of this and other defense-related genes to avoid resistance responses of the host plant. Enhanced resistance of RAP2.6 overexpression lines seemed to be due to enhanced callose deposition at syncytia which might interfere with nutrient import into syncytia.

Comparison of periplasmic and intracellular expression of Arabidopsis thionin proproteins in E. coli.

Thionins are antimicrobial plant peptides produced as preproproteins consisting of a signal peptide, the thionin domain, and a so-called acidic domain. Only thionin itself has been isolated from plants. To study the processing of the precursor, it has to be produced in a heterologous system. Since both domains contain several cysteines and, due to the known antimicrobial activity of the thionin, we tested the expression of all four Arabidopsis proproteins as fusion proteins. Periplasmic expression as fusion with maltose binding protein was not successful but cytoplasmic expression as His-tagged TRX fusion proteins with a TEV recognition sequence resulted in proteins of correct size. Use of the SHuffle strain C3030 further improved the expression. Fusion proteins inhibited growth of Escherichia coli. They could be cleaved by TEV protease, releasing authentic proproteins without any additional amino acid at the N-terminus.

Effect of green synthesized cerium oxide nanoparticles on fungal disease of wheat plants: A field study.

Recently, there has been considerable attention towards the production of environmentally friendly nanoparticles (NPs). In this investigation, the successful synthesis of cerium oxide nanoparticles (CeO2 NPs) was achieved by employing an eco-friendly technique that utilized an extract from the leaves of local plant quinoa (Chenopodium quinoa L.). The synthesized CeO2 NPs were subjected to characterization using state-of-the-art methods. The prepared CeO2 NPs contained a round shape with clusters and have a size of 7-10 nm. To assess how effective CeO2 NPs derived from C. quinoa were against Ustilago tritici, a fungal disease that negatively affects wheat crop globally, a study was performed on two varieties of wheat crop comprised of Arooj (V1) and Akber (V2), cultivated under field conditions. CeO2 NPs were applied foliarly twice to the wheat crop at four different concentrations: T0 (0 mg/L), T1 (50 mg/L), T2 (75 mg/L), and T3 (100 mg/L). The results revealed that the control group (T0) exhibited the highest disease severity index (DSI) with a value of 75% compared to the other concentrations of CeO2 NPs on both varieties. At a concentration of 100 mg/L of CeO2 NPs, the DSI dropped to a minimum of 35% and 37% on both V1 and V2 respectively. These findings indicated that an increase in the concentration of CeO2 NPs has a beneficial impact on disease severity. Similar patterns have also been observed with disease incidence (DI), with the greatest efficacy observed at a concentration of 100 mg/L of CeO2 NPs. Our investigation has shown that CeO2 NPs exhibitd significant antifungal potential against U. tritici which may be a promising strategy to mitigate fungal disease and crop losses globally.

Characterization of an Arabidopsis Defensin-like Gene Conferring Resistance against Nematodes.

Arabidopsis contains 317 genes for defensin-like (DEFL) peptides. DEFLs have been grouped into different families based mainly on cysteine motifs. The DEFL0770 group contains seven genes, of which four are strongly expressed in roots. We found that the expression of these genes is downregulated in syncytia induced by the beet cyst nematode Heterodera schachtii as revealed by RNAseq analysis. We have studied one gene of this group, At3g59930, in detail. A promoter::GUS line revealed that the gene is only expressed in roots but not in other plant organs. Infection of the GUS line with larvae of H. schachtii showed a strong downregulation of GUS expression in infection sites as early as 1 dpi, confirming the RNAseq data. The At3g59930 peptide had only weak antimicrobial activity against Botrytis cinerea. Overexpression lines had no enhanced resistance against this fungus but were more resistant to H. schachtii infection. Our data indicate that At3g59930 is involved in resistance to nematodes which is probably not due to direct nematicidal activity.

Analysis of a gene family for PDF-like peptides from Arabidopsis.

Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern which is stabilized by four disulfide bridges. We show here that Arabidopsis contains in addition to the proper plant defensins a group of 9 plant defensin-like (PdfL) genes. They are all expressed at low levels while GUS fusions of the promoters showed expression in most tissues with only minor differences. We produced two of the encoded peptides in E. coli and tested the antimicrobial activity in vitro. Both were highly active against fungi but had lower activity against bacteria. At higher concentrations hyperbranching and swollen tips, which are indicative of antimicrobial activity, were induced in Fusarium graminearum by both peptides. Overexpression lines for most PdfL genes were produced using the 35S CaMV promoter to study their possible in planta function. With the exception of PdfL4.1 these lines had enhanced resistance against F. oxysporum. All PDFL peptides were also transiently expressed in Nicotiana benthamiana leaves with agroinfiltration using the pPZP3425 vector. In case of PDFL1.4 this resulted in complete death of the infiltrated tissues after 7 days. All other PDFLs resulted only in various degrees of small necrotic lesions. In conclusion, our results show that at least some of the PdfL genes could function in plant resistance.

Transcription factors WRKY11 and WRKY17 are involved in abiotic stress responses in Arabidopsis.

Plant WRKY transcription factors play a vital role in abiotic stress tolerance and regulation of plant defense responses. This study examined AtWRKY11 and AtWRKY17 expression under ABA, salt, and osmotic stress at different developmental stages in Arabidopsis. We used reverse transcriptase PCR, quantitative real-time PCR, and promoter:GUS lines to analyze expression. Both genes were upregulated in response to abiotic stress. Next, we applied the same stressors to seedlings of T-DNA insertion wrky11 and 17 knock-out mutants (single and double). Under stress, the mutants exhibited slower germination and compromised root growth compared with the wild type. In most cases, double-mutant seedlings were more affected than single mutants. These results suggest that wrky11 and wrky17 are not strictly limited to plant defense responses but are also involved in conferring stress tolerance.

Transgenic Strategies for Enhancement of Nematode Resistance in Plants.

Plant parasitic nematodes (PPNs) are obligate biotrophic parasites causing serious damage and reduction in crop yields. Several economically important genera parasitize various crop plants. The root-knot, root lesion, and cyst nematodes are the three most economically damaging genera of PPNs on crops within the family Heteroderidae. It is very important to devise various management strategies against PPNs in economically important crop plants. Genetic engineering has proven a promising tool for the development of biotic and abiotic stress tolerance in crop plants. Additionally, the genetic engineering leading to transgenic plants harboring nematode resistance genes has demonstrated its significance in the field of plant nematology. Here, we have discussed the use of genetic engineering for the development of nematode resistance in plants. This review article also provides a detailed account of transgenic strategies for the resistance against PPNs. The strategies include natural resistance genes, cloning of proteinase inhibitor coding genes, anti-nematodal proteins and use of RNA interference to suppress nematode effectors. Furthermore, the manipulation of expression levels of genes induced and suppressed by nematodes has also been suggested as an innovative approach for inducing nematode resistance in plants. The information in this article will provide an array of possibilities to engineer resistance against PPNs in different crop plants.