The Compromised Recognition of Turnip Crinkle Virus1 Subfamily of Microrchidia ATPases Regulates Disease Resistance in Barley to Biotrophic and Necrotrophic Pathogens

MORC1 and MORC2, two of the seven members of the Arabidopsis (Arabidopsis thaliana) Compromised Recognition of Turnip Crinkle Virus1 subfamily of microrchidia Gyrase, Heat Shock Protein90, Histidine Kinase, MutL (GHKL) ATPases, were previously shown to be required in multiple layers of plant immunity. Here, we show that the barley (Hordeum vulgare) MORCs also are involved in disease resistance. Genome-wide analyses identified five MORCs that are 37% to 48% identical on the protein level to AtMORC1. Unexpectedly, and in clear contrast to Arabidopsis, RNA interference-mediated knockdown of MORC in barley resulted in enhanced basal resistance and effector-triggered, powdery mildew resistance locus A12-mediated resistance against the biotrophic powdery mildew fungus (Blumeria graminis f. sp. hordei), while MORC overexpression decreased resistance. Moreover, barley knockdown mutants also showed higher resistance to Fusarium graminearum. Barley MORCs, like their Arabidopsis homologs, contain the highly conserved GHKL ATPase and S5 domains, which identify them as members of the MORC superfamily. Like AtMORC1, barley MORC1 (HvMORC1) binds DNA and has Mn²⁺-dependent endonuclease activities, suggesting that the contrasting function of MORC1 homologs in barley versus Arabidopsis is not due to differences in their enzyme activities. In contrast to AtMORCs, which are involved in silencing of transposons that are largely restricted to pericentromeric regions, barley MORC mutants did not show a loss-of-transposon silencing regardless of their genomic location. Reciprocal overexpression of MORC1 homologs in barley and Arabidopsis showed that AtMORC1 and HvMORC1 could not restore each other’s function. Together, these results suggest that MORC proteins function as modulators of immunity, which can act negatively (barley) or positively (Arabidopsis) dependent on the species.The evolution of a complex defense system has been the consequence of plants being constantly exposed to pathogenic microbes and pests. One of the first lines of active defense is based on a perception of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors located in the plant cell membrane. The defense response to PAMP recognition is called PAMP-triggered immunity (PTI). While PTI is sufficient to stop colonization by many microbes, some microorganisms overcome this immune response by releasing effectors (formerly called virulence factors). In a coevolutionary process, some plants have evolved resistance (R) proteins for direct or indirect recognition of microbial effectors (avirulence [Avr] factors) leading to effector-triggered immunity (ETI). ETI is frequently characterized by a rapid and locally restricted programmed cell death response (also known as hypersensitive reaction [HR]), which helps to limit pathogen proliferation and disease symptoms. On the contrary, the absence of an Avr-R protein interaction results in virulence of the pathogen. In addition, ETI is counteracted by some microbes by the release of additional virulence factors that block or overcome effector recognition and ensure pathogenicity. The mutual evolution of host and microbe leading to elicitation or suppression of ETI is summarized by the “zigzag” model proposed by Jones and Dangl (2006). PTI and ETI are associated with activation of various defense responses both at infection sites and in distal tissue, including production and accumulation of reactive oxygen species, salicylic acid, and pathogenesis-related proteins. Systemic activation of such responses, triggered in the uninfected tissue, leads to long-lasting, broad-based resistance to subsequent pathogen infections, termed systemic acquired resistance.A genetic screen in Arabidopsis (Arabidopsis thaliana) searching for mutants with compromised resistance mediated by the R protein HR to Turnip Crinkle Virus (HRT) against Turnip Crinkle Virus (TCV) led to the discovery of the Compromised Recognition of TCV1 (CRT1) subfamily of the microrchidia (MORC) subclade of the GHKL (for Gyrase, Heat Shock Protein90, Histidine Kinase, MutL) ATPase superfamily (Watson et al., 1998; Iyer et al., 2008; Kang et al., 2008). Genome analysis of Arabidopsis revealed that MORC1 (formerly named CRT1 in Kang et al., 2008, 2010, 2012) has two close (>70% sequence similarity on amino acid [aa] level) and four distant (<50% aa similarity) homologs. A double knockout mutant, morc1-2 morc2-1, lacking MORC1 and its closest homolog MORC2 also displayed compromised ETI to avirulent Pseudomonas syringae, suppressed basal resistance, systemic acquired resistance, and/or PTI to TCV and virulent P. syringae and compromised nonhost resistance to Phytophthora infestans (Kang et al., 2012). Arabidopsis MORC1 physically interacts with at least eleven R proteins belonging to three different structural classes (Martin et al., 2003), including HRT, the R protein involved in recognition of TCV. This interaction is a dynamic process, as MORC1 bound inactive R proteins, while little or no interaction was observed when the R proteins were activated (Kang et al., 2010). Taken together, these results argued that MORC1 protein family members in Arabidopsis are key components in multiple layers of resistance against a variety of pathogens. Recently, it was shown that a small fraction of AtMORC1 translocates to the plant nucleus after ETI and PTI activation (Kang et al., 2012). Because Arabidopsis MORC1 possesses DNA/RNA-binding capacity and endonuclease activity in vitro, these findings suggest a potential role in DNA recombination and repair (Kang et al., 2012). In addition, three recent independent studies identified Arabidopsis MORC1 and its homolog MORC6 (also named Defective in Meristem Silencing11) as novel factors involved in gene silencing and/or chromatin superstructure remodeling in response to epigenetic signals (Lorković et al., 2012; Moissiard et al., 2012; Brabbs et al., 2013).Given that the CRT1 subfamily of MORC ATPases is involved in multiple layers of disease resistance against various pathogens, these genes may have relevance for agronomic applications. To assess whether MORCs are involved in crop plant resistance and thus could be exploited in breeding strategies, MORC1 homologous genes were identified in the model cereal crop barley (Hordeum vulgare). We show here that all five barley MORCs, discovered in the not yet fully annotated barley genome, are involved in resistance to agronomically important diseases. Unexpectedly, however, and in clear contrast to Arabidopsis, barley plants silenced for MORC genes were more resistant, while overexpression compromised resistance to infections by both biotrophic and necrotrophic fungal pathogens. Moreover, reciprocal overexpression in Arabidopsis and barley showed that AtMORC1 and HvMORC1 homologs are not functionally interchangeable.     

Ethnobotanical study and conservation status of trees in the district Sargodha,Punjab, Pakistan

Sargodha district is one of the least studied regions of Pakistan regarding its ethnobotanical values. This paper is the first report related to the documentation and conservation status of the tree species in the Sargodha district, and their folk ethnobotanical uses. An interview base survey was conducted in the study area in 2010-2013. The ethnobotanical data revealed the use of 100 tree species (6 gymnosperms, 94 angiosperms) belonging to 77 genera (6 gymnosperms, 71 angiosperms) and 39 families (4 gymnosperms, 35 angiosperms), with the Fabaceae ranking first with 19 tree species, followed by the Moraceae (12 species). Tree species like Aegle marmelos, Butea monosperma, Diospyrus malabarica, Gmelina arborea, Kigelia africana, Manilkara hexandra, Manilkara zapota, Mimusops elengi, Nyctanthes arbor-tristis, Putranjiva roxburghii, Terminalia arjuna and Terminalia bellerica are not only unique in their medicinal value but also interesting because of their unusual occurrence here. Thevetia peruviana, Cassia fistula, Celtis australis, Delonix regia, Diospyrus malabarica, Grevillea robusta, Haplophragma adenophylum, Jacaranda mimosifolia, Lagerstroemia speciosa, Plumeria rubra, Pterospermum acerifolium, Roystonea regia, Taxodium distichum and Tectona grandis are included among the worth looking ornamental tree species. Capparis decidua, Dalbergia sissoo, Tamarix aphylla, Tamarix dioica, Prosopis cineraria and Ziziphus mauritiana are the most commonly used timber species. Other common ethnobotanical utilization of these trees includes either sheltering or fuel or agricultural uses. Lack of awareness about the potential uses of these species, and particularly ignorance of the concerned authorities, have led to a decline in the population of this precious tree flora. Documentation of this tree flora, and as-sociated indigenous knowledge, can be used as a basis for developing management plans for conservation and sustainable use of this flora in the study area. A well-organized management is critical to restore and conserve this endangered natural resource in the District Sargodha, Pakistan. The immense medicinal and timber value of these tree species make it necessary to promote their conservation to simultaneously alleviate the poverty and improve the socio-economic status of the study area.     

Interactive signal transfer between host and pathogen during successful infection of barley leaves by Blumeria graminis and Bipolaris sorokiniana

Using ion-selective microprobes, interactive signalling between barley and Blumeria graminis or Bipolaris sorokiniana has been investigated. The question was raised whether a biotrophically growing fungus manipulates the electrical driving forces (membrane potential, transmembrane pH), required for H⁺ cotransport of energy-rich compounds. Electrodes were positioned in the substomatal cavity of open stomata or on the leaf surface, and pH was measured continuously up to several days during fungal development. We demonstrate that surface and apoplastic fluids are electrically coupled and respond in a similar manner to stimuli. Apoplastic pH, monitored from the moment of inoculation with conidia, reveals several phases: 2–4 h after inoculation of the barley leaf with either fungus, the host displays rapid transient responses after its first contact with the fungal cell wall; apoplastic pH and pCa increases, cytoplasmic pH and pCa decreases. About 1 day after inoculation, the apoplastic pH increases by up to 2 pH units, which is thought to reflect a resistance response against the intruder. Whereas barley leaf cells possess a membrane potential of −152±5 mV, hyphae of B. graminis yield −251±8 mV, indicative of a substantial driving force advantage for the fungus. Although the resting membrane potential of barley remains constant during the first days after inoculation, leaves infected with B. sorokiniana get confronted with an energy problem, indicated by a retarded repolarization following a “light-off” stimulus. Five days after inoculation, apoplastic pH has increased to 5.97±0.47 (n=11) and does no longer respond to “light-off” when measured within lesions. In contrast, it stays at near normal values outside the lesions and responds to “light-off”.It is concluded that biotrophically growing fungi do not manipulate the cotransport driving forces since (i) any change in apoplastic pH would be experienced by both partners; (ii) the resting membrane potential is not changed. It is suggested that measured pH changes reflect defence responses of the host against the fungus rather than fungal action to increase compatibility.     

NPR1 is required for root colonization and the establishment of a mutualistic symbiosis between the beneficial bacterium Rhizobium radiobacter and barley

Non-expressor of pathogenesis-related genes 1 (NPR1) is a key regulator of plant innate immunity and systemic disease resistance. The model for NPR1 function is based on experimental evidence obtained largely from dicots; however, this model does not fit all aspects of Poaceae family, which includes major crops such as wheat, rice and barley. In addition, there is little scientific data on NPR1's role in mutualistic symbioses. We assessed barley (Hordeum vulgare) HvNPR1 requirement during the establishment of mutualistic symbiosis between barley and beneficial Alphaproteobacterium Rhizobium radiobacter F4 (RrF4). Upon RrF4 root-inoculation, barley NPR1-knockdown (KD-hvnpr1) plants lost the typical spatiotemporal colonization pattern and supported less bacterial multiplication. Following RrF4 colonization, expression of salicylic acid marker genes were strongly enhanced in wild-type roots; whereas in comparison, KD-hvnpr1 roots exhibited little to no induction. Both basal and RrF4-induced root-initiated systemic resistance against virulent Blumeria graminis were impaired in leaves of KD-hvnpr1. Besides these immune-related differences, KD-hvnpr1 plants displayed higher root and shoot biomass than WT. However, RrF4-mediated growth promotion was largely compromised in KD-hvnpr1. Our results demonstrate a critical role for HvNPR1 in establishing a mutualistic symbiosis between a beneficial bacterium and a cereal crop.     

Flower visitation and oviposition behavior of Frankliniella occidentalis (Tysan., Thripidae) on cucumber plants

Oviposition behaviour of western flower thrips, Frankliniella occidentalis (Pergande) on greenhouse cucumber, Cucumis satifus (L.) was investigated. Most eggs were laid in the leaves, along veins and under leaf hairs, with only a few on plant stems and flowers. Oviposition rate was higher during the day than during the night. During the day, more adult thrips were found in the flowers than during the night. The number of adult thrips per flower increased rapidly after sunrise with the highest densities occurring around noon and thereafter the number of thrips in flowers decreased during the afternoon. No differences were found in the number of larvae (first and second instars) in flowers during the same period. The number of adult thrips on male and female cucumber flowers was not different, indicating that pollen is not the only attraction in flowers for thrips.