Abdelnaser Elashry

A parasitic nematode releases cytokinin that controls cell division and orchestrates feeding site formation in host plants

Significance Sedentary plant-parasitic cyst nematodes are microscopic roundworms that cause significant yield losses in agriculture. Successful parasitism is based on the formation of a hypermetabolic feeding site in host roots from which the nematodes withdraw their nutrients. The host cell cycle is activated at the site of infection and contributes to the formation of the syncytium. Here, we provide genetic evidence that nematode-derived cytokinin is involved in activating the host cell cycle during infection. Our findings show the ability of an animal to synthesize and secrete a functional plant hormone to establish long-term parasitism. Sedentary plant-parasitic cyst nematodes are biotrophs that cause significant losses in agriculture. Parasitism is based on modifications of host root cells that lead to the formation of a hypermetabolic feeding site (a syncytium) from which nematodes withdraw nutrients. The host cell cycle is activated in an initial cell selected by the nematode for feeding, followed by activation of neighboring cells and subsequent expansion of feeding site through fusion of hundreds of cells. It is generally assumed that nematodes manipulate production and signaling of the plant hormone cytokinin to activate cell division. In fact, nematodes have been shown to produce cytokinin in vitro; however, whether the hormone is secreted into host plants and plays a role in parasitism remained unknown. Here, we analyzed the spatiotemporal activation of cytokinin signaling during interaction between the cyst nematode, Heterodera schachtii, and Arabidopsis using cytokinin-responsive promoter:reporter lines. Our results showed that cytokinin signaling is activated not only in the syncytium but also in neighboring cells to be incorporated into syncytium. An analysis of nematode infection on mutants that are deficient in cytokinin or cytokinin signaling revealed a significant decrease in susceptibility of these plants to nematodes. Further, we identified a cytokinin-synthesizing isopentenyltransferase gene in H. schachtii and show that silencing of this gene in nematodes leads to a significant decrease in virulence due to a reduced expansion of feeding sites. Our findings demonstrate the ability of a plant-parasitic nematode to synthesize a functional plant hormone to manipulate the host system and establish a long-term parasitic interaction.

Next Generation Sequencing Based Transcriptome Analysis of Septic-Injury Responsive Genes in the Beetle Tribolium castaneum

Beetles (Coleoptera) are the most diverse animal group on earth and interact with numerous symbiotic or pathogenic microbes in their environments. The red flour beetle Tribolium castaneum is a genetically tractable model beetle species and its whole genome sequence has recently been determined. To advance our understanding of the molecular basis of beetle immunity here we analyzed the whole transcriptome of T. castaneum by high-throughput next generation sequencing technology. Here, we demonstrate that the Illumina/Solexa sequencing approach of cDNA samples from T. castaneum including over 9.7 million reads with 72 base pairs (bp) length (approximately 700 million bp sequence information with about 30× transcriptome coverage) confirms the expression of most predicted genes and enabled subsequent qualitative and quantitative transcriptome analysis. This approach recapitulates our recent quantitative real-time PCR studies of immune-challenged and naïve T. castaneum beetles, validating our approach. Furthermore, this sequencing analysis resulted in the identification of 73 differentially expressed genes upon immune-challenge with statistical significance by comparing expression data to calculated values derived by fitting to generalized linear models. We identified up regulation of diverse immune-related genes (e.g. Toll receptor, serine proteinases, DOPA decarboxylase and thaumatin) and of numerous genes encoding proteins with yet unknown functions. Of note, septic-injury resulted also in the elevated expression of genes encoding heat-shock proteins or cytochrome P450s supporting the view that there is crosstalk between immune and stress responses in T. castaneum. The present study provides a first comprehensive overview of septic-injury responsive genes in T. castaneum beetles. Identified genes advance our understanding of T. castaneum specific gene expression alteration upon immune-challenge in particular and may help to understand beetle immunity in general.

An Arabidopsis ATPase gene involved in nematode-induced syncytium development and abiotic stress responses

The beet cyst nematode Heterodera schachtii induces syncytia in the roots of Arabidopsis thaliana, which are its only nutrient source. One gene, At1g64110, that is strongly up-regulated in syncytia as shown by RT-PCR, quantitative RT-PCR, in situ RT-PCR and promoter::GUS lines, encodes an AAA+-type ATPase. Expression of two related genes in syncytia, At4g28000 and At5g52882, was not detected or not different from control root segments. Using amiRNA lines and T-DNA mutants, we show that At1g64110 is important for syncytium and nematode development. At1g64110 was also inducible by wounding, jasmonic acid, salicylic acid, heat and cold, as well as drought, sodium chloride, abscisic acid and mannitol, indicating involvement of this gene in abiotic stress responses. We confirmed this using two T-DNA mutants that were more sensitive to abscisic acid and sodium chloride during seed germination and root growth. These mutants also developed significantly smaller roots in response to abscisic acid and sodium chloride. An in silico analysis showed that ATPase At1g64110 (and also At4g28000 and At5g52882) belong to the ‘meiotic clade’ of AAA proteins that includes proteins such as Vps4, katanin, spastin and MSP1.

The AAP gene family for amino acid permeases contributes to development of the cyst nematode Heterodera schachtii in roots of Arabidopsis

The beet cyst nematode Heterodera schachtii is able to infect Arabidopsis plants and induce feeding sites in the root. These syncytia are the only source of nutrients for the nematodes throughout their life and are a nutrient sink for the host plant. We have studied here the role of amino acid transporters for nematode development. Arabidopsis contains a large number of different amino acid transporters in several gene families but those of the AAP family were found to be especially expressed in syncytia. Arabidopsis contains 8 AAP genes and they were all strongly expressed in syncytia with the exception of AAP5 and AAP7, which were slightly downregulated. We used promoter::GUS lines and in situ RT-PCR to confirm the expression of several AAP genes and LHT1, a lysine- and histidine-specific amino acid transporter, in syncytia. The strong expression of AAP genes in syncytia indicated that these transporters are important for the transport of amino acids into syncytia and we used T-DNA mutants for several AAP genes to test for their influence on nematode development. We found that mutants of AAP1, AAP2, and AAP8 significantly reduced the number of female nematodes developing on these plants. Our study showed that amino acid transport into syncytia is important for the development of the nematodes.

A distinct role of pectate lyases in the formation of feeding structures induced by cyst and root-knot nematodes.

Pectin in the primary plant cell wall is thought to be responsible for its porosity, charge density, and microfibril spacing and is the main component of the middle lamella. Plant-parasitic nematodes secrete cell wall-degrading enzymes that macerate the plant tissue, facilitating the penetration and migration within the roots. In sedentary endoparasitic nematodes, these enzymes are released only during the migration of infective juveniles through the root. Later, nematodes manipulate the expression of host plant genes, including various cell wall enzymes, in order to induce specific feeding sites. In this study, we investigated expression of two Arabidopsis pectate lyase-like genes (PLL), PLL18 (At3g27400) and PLL19 (At4g24780), together with pectic epitopes with different degrees of methylesterification in both syncytia induced by the cyst nematode Heterodera schachtii and giant cells induced by the root-knot nematode Meloidogyne incognita. We confirmed upregulation of PLL18 and PLL19 in both types of feeding sites with quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) and in situ RT-PCR. Furthermore, the functional analysis of mutants demonstrated the important role of both PLL genes in the development and maintenance of syncytia but not giant cells. Our results show that both enzymes play distinct roles in different infected root tissues as well as during parasitism of different nematodes.

Identification and characterisation of resistance to the cereal cyst nematode Heterodera filipjevi in winter wheat

The aim of this study was to search for new sources of resistance against the cereal cyst nematode, Heterodera filipjevi , in a collection of 290 wheat accessions. The plants were inoculated with juveniles and assessed for the number of females and cysts. One percent of the wheat accessions were ranked as resistant, 16% as moderately resistant, 41% as moderately susceptible, 26% as susceptible and 15% as highly susceptible. The infection rate and the number of females and cysts per plant were significantly lower in the resistant accession Nudakota and three moderately resistant accessions Ekonomka, Katea and Lantian 12 compared with susceptible cv. Bezostaya 1. Nematode development was reduced in resistant and moderately resistant accessions. The size of females and the total number of eggs and second-stage juveniles were reduced only in Ekonomka. No significant difference in plant height, plant weight, root length, root weight and root volume were recorded for inoculated plants compared to non-inoculated plants. This study has identified four resistant wheat accessions offering new material for breeding the resistance to H. filipjevi .

Heterodera schachtii Tyrosinase-like protein - a novel nematode effector modulating plant hormone homeostasis

The beet cyst nematode Heterodera schachtii causes major yield losses in sugar beet. Understanding the interaction between H. schachtii and its host plant is important for developing a sustainable management system. Nematode effectors play a crucial role in initializing and sustaining successful parasitism. In our study, we identified a gene (Hs-Tyr) encoding a tyrosinase functional domain (PF00264). We describe Hs-Tyr as a novel nematode effector. Hs-Tyr is localized in the nematode esophageal gland. Up-regulation of its expression coincided with the parasitic developmental stages of the nematode. Silencing Hs-Tyr by RNA interference made the treated nematodes less virulent. When RNAi-treated nematodes succeeded in infecting the plant, developing females and their associated syncytial nurse cells were significantly smaller than in control plants. Ectopically expressing the Hs-Tyr effector in Arabidopsis increased plant susceptibility to H. schachtii, but not to the root-knot nematode Meloidogyne incognita. Interestingly, Hs-Tyr in the plant promoted plant growth and changed the root architecture. Additionally, the expression of Hs-Tyr in Arabidopsis caused changes in the homeostasis of several plant hormones especially auxin and the ethylene precursor aminocyclopropane-carboxylic acid.

Genome-Wide Association Study in Wheat Identifies Resistance to the Cereal Cyst Nematode Heterodera filipjevi.

The cyst nematode Heterodera filipjevi is a plant parasite causing substantial yield loss in wheat. Resistant cultivars are the preferred method of controlling cyst nematodes. Association mapping is a powerful approach to detect associations between phenotypic variation and genetic polymorphisms; in this way favorable traits such as resistance to pathogens can be located. Therefore, a genome-wide association study of 161 winter wheat accessions was performed with a 90K iSelect single nucleotide polymorphism (SNP) chip. Population structure analysis grouped into two major subgroups and first principal component accounted 6.16% for phenotypic diversity. The genome-wide linkage disequilibrium across wheat was 3 cM. Eleven quantitative trait loci (QTLs) on chromosomes 1AL, 2AS, 2BL, 3AL, 3BL, 4AS, 4AL, 5BL, and 7BL were identified using a mixed linear model false discovery rate of P < 0.01 that explained 43% of total genetic variation. This is the first report of QTLs conferring resistance to H. filipjevi in wheat. Eight QTLs on chromosomes 1AL, 2AS, 2BL, 3AL, 4AL, and 5BL were linked to putative genes known to be involved in plant-pathogen interactions. Two other QTLs on 3BL and one QTL on 7BL linked to putative genes known to be involved in abiotic stress.

Identification and characterization of a putative protein disulfide isomerase (HsPDI) as an alleged effector of Heterodera schachtii

The plant-parasitic nematode Heterodera schachtii is an obligate biotroph that induces syncytial feeding sites in roots of its hosts. Nematodes produce effectors that are secreted into the host and facilitate infection process. Here we identified H. schachtii protein disulphide isomerase (HsPDI) as a putative effector that interferes with the host’s redox status. In situ hybridization showed that HsPdi is specifically localized within esophageal glands of pre-parasitic second stage juveniles (J2). HsPdi is up-regulated in the early parasitic J2s. Silencing of HsPdi by RNA interference in the J2s hampers their development and leads to structural malfunctions in associated feeding sites induced in Arabidopsis roots. Expression of HsPDI in Arabidopsis increases plant’s susceptibility towards H. schachtii. HsPdi expression is up-regulated in the presence of exogenous H2O2, whereas HsPdi silencing results in increased mortality under H2O2 stress. Stable expression of HsPDI in Arabidopsis plants decreases ROS burst induced by flg22. Transiently expressed HsPDI in N. benthamiana leaves is localized in the apoplast. HsPDI plays an important role in the interaction between nematode and plant, probably through inducing local changes in the redox status of infected host tissue. It also contributes to protect the nematode from exogenous H2O2 stress.

Identification of Two Meloidogyne hapla Genes and an Investigation of Their Roles in the Plant-Nematode Interaction

Root-knot nematodes are soil-borne pathogens that invade and establish feeding sites in plant roots. They have an extremely broad host range, including most vascular plants. During infection of a susceptible host, root-knot nematodes secrete molecules called effectors that help them establish an intimate interaction with the plant and, at the same time, allow them to evade or suppress plant immune responses. Despite the fact that Meloidogyne hapla is a significant pest on several food crops, no effectors have been characterized from this root-knot nematode species thus far. Using the published genome and proteome from M. hapla, we have identified and characterized two genes, MhTTL2 and Mh265. MhTTL2 encodes a predicted secreted protein containing a transthyretin-like protein domain. The expression of MhTTL2 was up-regulated during parasitic life stages of the nematode, and in situ hybridization showed that MhTTL2 was expressed in the amphids, suggesting it has a role in the nematode nervous system during parasitism. We also studied the gene Mh265. The Mh265 transcript was localized to the subventral esophageal glands. An upregulation in Mh265 expression coincided with the pre- and early-parasitic life stages of the nematode. When Mh265 was constitutively expressed in plants, it enhanced their susceptibility to nematodes. These transgenic plants were also compromised in flg22-induced callose deposition, suggesting the Mh265 is modulating plant basal immune responses.