Amjad Abbas

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

BackgroundCyst 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.ResultsExpression 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.ConclusionsOur 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.

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.

GENOME-WIDE ANALYSIS OF TRIHELIX TRANSCRIPTION FACTOR GENE FAMILY IN Arabidopsis thaliana

Transcriptional factors are the proteins that play an important role in the gene regulation by activating or repressing transcription of downstream target genes and consequently controlling many cellular activities during plant growth and development (Gao et al., 2013; Ling et al., 2011). This is achieved when DNA binding domain of transcriptional factors bind with specific sequences called cis-acting elements present in the promoters of the genes being regulated (Nuruzzaman et al., 2012). The plants have more than 60 families of transcription factors with different functional activities (Kaplan-Levy et al., 2012; Qin et al., 2014). In plants, the transcriptional factors are involved in many processes i.e. plant growth and development as well as regulation of abiotic and biotic stress responses of the plants (Osorio et al., 2012; Xie et al., 2009). This paper discusses about analysis of trihelix transcription factor gene family in Arabidopsis. The DNA-binding proteins characterized by the trihelix motif are solely present in plants. They were discovered in 1990s and are one of the first transcription factor gene family found in plants known as GT factors because of their binding properties with GT elements (Kaplan-Levy et al., 2012; Qin et al., 2014). Trihelix domain attracted the scientists because it is the only class of three spiro spin structure; it contains three tandem repeats helix loop helix loop helix (Luo et al., 2012). The history of trihelix revealed that they were first identified in pea (Pisum sativum) nucleus later in soybean (Glycine max Merr.), tobacco (Nicotiana tabacum L.), rice (oryza sativa), and Arabidopsis (Luo et al. 2012). Although the trihelix family is confined to terrestrial plants but their existence in humans, animals and Drosophila needs investigation (Riaño-Pachón et al., 2008). They are not present in the green algae (Chlorophyta) and have undergone massive expansion in the lineage of the common ascendant of terrestrial plants (Lang et al., 2010). In the initial studies, the member of the gene family were thought to be involved in light-responsive gene regulation but the later studies highlighted their involvement in growth, development of tissues, embryo development, petal loss and plant organs, in biotic and abiotic stresses. Various genes of this family show variety of functions in plants like AT5G03680 (PTL) (Petal loss) gene is involved in morphological activities of flower organs (Kaplan-Levy et al., 2012), AT1G54060 (ASIL1) and AT3G14180 (ASIL2) are reported to be involved in chlorophyll accumulation during embryo development and GT-1 is involved in light responses (Qin et al., 2014). Trihelix factors bind to motifs called GT elements on the promoter DNA but the trihelix motif is only confined to the GT-1 and GT-2 DNA-binding proteins. Their genomic studies, functional studies and the structural pattern Pak. J. Agri. Sci., Vol. 53(2), 439-448; 2016 ISSN (Print) 0552-9034, ISSN (Online) 2076-0906 DOI: 10.21162/PAKJAS/16.3347 http://www.pakjas.com.pk

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.

The Good, the Bad, and the Ugly of Rhizosphere Microbiome

Rhizosphere is the portion of soil that is exposed to the root activity. It is hot spot for microbial activities which support the plant growth and development in different ways. Microbial communities in the rhizosphere referred as rhizosphere microbiome are one of the most diverse regions of the ecosystem existing on Earth. Rhizosphere microbiome is biologically the most diverse part of the ecosystem which contains a large number of microbial communities which interact with the plants differently like the good, the bad, and the ugly microbes of rhizosphere. The good ones are beneficial microbes of the rhizosphere which are involved in plant growth promotion through nutrient uptake in plants, antagonism to plant pathogens, and plant tolerance against abiotic stresses. However, the bad ones are plant parasitic fungi and nematodes which cause diseases of economic importance in important crop plants and result in serious issues of reduction in productivity and food security. Similarly, some rhizosphere microbes avail the opportunity to invade the human body through different courses and cause infectious diseases. These opportunistic microbes are “the ugly” ones as they are the most deleterious in nature. In this chapter, we have discussed in detail the good, the bad, and the ugly members of rhizosphere microbiome. Moreover, we have given a comprehensive account of bolts and nuts of rhizosphere and engineering of rhizosphere for agriculturally sustainability.