Overexpression of VIRE2-INTERACTING PROTEIN2 in Arabidopsis regulates genes involved in Agrobacterium-mediated plant transformation and abiotic stresses.

Arabidopsis VIRE2-INTERACTING PROTEIN2 (VIP2) was previously described as a protein with a NOT domain, and Arabidopsis vip2 mutants are recalcitrant to Agrobacterium-mediated root transformation. Here we show that VIP2 is a transcription regulator and the C-terminal NOT2 domain of VIP2 interacts with VirE2. Interestingly, AtVIP2 overexpressor lines in Arabidopsis did not show an improvement in Agrobacterium-mediated stable root transformation, but the transcriptome analysis identified 1,634 differentially expressed genes compared to wild-type. These differentially expressed genes belonged to various functional categories such as membrane proteins, circadian rhythm, signaling, response to stimulus, regulation of plant hypersensitive response, sequence-specific DNA binding transcription factor activity and transcription regulatory region binding. In addition to regulating genes involved in Agrobacterium-mediated plant transformation, AtVIP2 overexpressor line showed differential expression of genes involved in abiotic stresses. The majority of the genes involved in abscisic acid (ABA) response pathway, containing the Abscisic Acid Responsive Element (ABRE) element within their promoters, were down-regulated in AtVIP2 overexpressor lines. Consistent with this observation, AtVIP2 overexpressor lines were more susceptible to ABA and other abiotic stresses. Based on the above findings, we hypothesize that VIP2 not only plays a role in Agrobacterium-mediated plant transformation but also acts as a general transcriptional regulator in plants.

Agrobacterium may delay plant nonhomologous end-joining DNA repair via XRCC4 to favor T-DNA integration.

Agrobacterium tumefaciens is a soilborne pathogen that causes crown gall disease in many dicotyledonous plants by transfer of a portion of its tumor-inducing plasmid (T-DNA) into the plant genome. Several plant factors that play a role in Agrobacterium attachment to plant cells and transport of T-DNA to the nucleus have been identified, but the T-DNA integration step during transformation is poorly understood and has been proposed to occur via nonhomologous end-joining (NHEJ)-mediated double-strand DNA break (DSB) repair. Here, we report a negative role of X-ray cross complementation group4 (XRCC4), one of the key proteins required for NHEJ, in Agrobacterium T-DNA integration. Downregulation of XRCC4 in Arabidopsis and Nicotiana benthamiana increased stable transformation due to increased T-DNA integration. Overexpression of XRCC4 in Arabidopsis decreased stable transformation due to decreased T-DNA integration. Interestingly, XRCC4 directly interacted with Agrobacterium protein VirE2 in a yeast two-hybrid system and in planta. VirE2-expressing Arabidopsis plants were more susceptible to the DNA damaging chemical bleomycin and showed increased stable transformation. We hypothesize that VirE2 titrates or excludes active XRCC4 protein available for DSB repair, thus delaying the closure of DSBs in the chromosome, providing greater opportunity for T-DNA to integrate.

Are communities of microbial symbionts more diverse than communities of macrobial hosts?

In this study, fungal viruses (mycoviruses) of plant-associated fungi were used to test the general assertion that communities of parasitic and mutualistic symbionts may be more species-diverse than communities of their hosts. Mycoviruses are poorly studied in general, but can affect the fitness and ecology of the fungi and plants with which they associate. To date, mycovirus incidence and diversity in natural communities remain largely unaddressed. Here, we compared the incidence and diversity of fungi associated with tallgrass prairie plants to the diversity and incidence of mycoviruses within those fungi. Specifically, we sampled viruses from fungi associated with a parasitic plant (Cuscuta cuspidata) and its most frequent host plant (Ambrosia psilostachya) in a tallgrass prairie habitat in Oklahoma. For each plant sample we cultured fungal endophytes from surface-sterilized above-ground tissues. From the cultured fungi we extracted DNA to identify fungi, and extracted double-stranded RNA (dsRNA) to detect mycoviruses. Mycoviruses were further characterized using reverse transcription-PCR and sequence analyses. We found at least 25 fungal taxa associated with the two plants, and 10 % of these fungi contained readily detectable viruses. Several mycovirus types were shared among fungal taxa, indicating that mycoviruses may be less specialized than originally thought. Although the virus community was not as diverse as the fungal endophyte community (16 taxa), species accumulation rates of mycoviruses (inferred from rescaled rarefaction curves) may be higher than those of their associated fungal hosts. Thus, mycoviruses represent a further layer of undocumented biodiversity in ecological communities.

Teasing apart a three-way symbiosis: transcriptome analyses of Curvularia protuberata in response to viral infection and heat stress.

The fungus Curvularia protuberata carries a dsRNA virus, Curvularia thermal tolerance virus, and develops a three-way symbiotic relationship with plants to enable their survival in extreme soil temperatures. To learn about the genome of C. protuberata and possible mechanisms of heat tolerance a collection of expressed sequence tags (ESTs) were developed from two subtracted cDNA libraries from mycelial cultures grown under control and heat stress conditions. We analyzed 4207 ESTs that were assembled into 1926 unique transcripts. Of the unique transcripts, 1347 (70%) had sequence similarity with GenBank entries using BLASTX while the rest represented unknown proteins with no matches in the databases. The majority of ESTs with known similarities were homologues to fungal genes. The EST collection presents a rich source of heat stress and viral induced genes of a fungal endophyte that is involved in a symbiotic relationship with plants. Expression profile analyses of some candidate genes suggest possible involvement of osmoprotectants such as trehalose, glycine betaine, and taurine in the heat stress response. The fungal pigment melanin, and heat shock proteins also may be involved in the thermotolerance of C. protuberata in culture. The results assist in understanding the molecular basis of thermotolerance of the three-way symbiosis. Further studies will confirm or refute the involvement of these pathways in stress tolerance.

The EAR-motif of the Cys2/His2-type zinc finger protein Zat7 plays a key role in the defense response of Arabidopsis to salinity stress.

Cys2/His2-type zinc finger proteins, which contain the EAR transcriptional repressor domain, are thought to play a key role in regulating the defense response of plants to biotic and abiotic stress conditions. Although constitutive expression of several of these proteins was shown to enhance the tolerance of transgenic plants to abiotic stress, it is not clear whether the EAR-motif of these proteins is involved in this function. In addition, it is not clear whether suppression of plant growth, induced in transgenic plants by different Cys2/His2 EAR-containing proteins, is mediated by the EAR-domain. Here we report that transgenic Arabidopsis plants constitutively expressing the Cys2/His2 zinc finger protein Zat7 have suppressed growth and are more tolerant to salinity stress. A deletion or a mutation of the EAR-motif of Zat7 abolishes salinity tolerance without affecting growth suppression. These results demonstrate that the EAR-motif of Zat7 is directly involved in enhancing the tolerance of transgenic plants to salinity stress. In contrast, the EAR-motif appears not to be involved in suppressing the growth of transgenic plants. Further analysis of Zat7 using RNAi lines suggests that Zat7 functions in Arabidopsis to suppress a repressor of defense responses. A yeast two-hybrid analysis identified putative interactors of Zat7 and the EAR-domain, including WRKY70 and HASTY, a protein involved in miRNA transport. Our findings demonstrate that the EAR-domain of Cys2/His2-type zinc finger proteins plays a key role in the defense response of Arabidopsis to abiotic stresses.

Alteration of oxidative and carbohydrate metabolism under abiotic stress in two rice (Oryza sativa L.) genotypes contrasting in chilling tolerance.

Abiotic stress is a major limiting factor in crop production. Physiological comparisons between contrasting abiotic stress-tolerant genotypes will improve understanding of stress-tolerant mechanisms. Rice seedlings (S3 stage) of a chilling-tolerant (CT) genotype (CT6748-8-CA-17) and a chilling-sensitive (CS) genotype (INIAP12) were subjected to abiotic stresses including chilling (13/12 degrees C), salt (100mM NaCl), and osmotic (200mM mannitol). Measures of physiological response to the stresses included changes in stress-related sugars, oxidative products and protective enzymes, parameters that could be used as possible markers for selection of improved tolerant varieties. Metabolite analyses showed that the two genotypes responded differently to different stresses. Genotype survival under chilling-stress was as expected, however, CT was more sensitive to salt stress than the CS genotype. The CT genotype was able to maintain membrane integrity better than CS, perhaps by reduction of lipid peroxidation via increased levels of antioxidant enzymes during chilling stress. This genotype accumulated sugars in response to stress, but the accumulation was usually less than in the CS genotype. Chill-stressed CT accumulated galactose and raffinose whereas these saccharides declined in CS. On the other hand, the tolerance mechanism in the more salt- and water-deficit-tolerant CS may be associated with accumulation of osmoprotectants such as glucose, trehalose and mannitol.

The OsLti6 genes encoding low-molecular-weight membrane proteins are differentially expressed in rice cultivars with contrasting sensitivity to low temperature.

Rice (Oryza sativa L.) is sensitive to chilling particularly at early stages of seedling establishment. Two closely related genes (OsLti6a, OsLti6b), which are induced by low temperature during seedling emergence were isolated from a cold tolerant temperate japonica rice cultivar. These genes are closely related to the Arabidopsis rare cold-inducible (RCI2) and barley low-temperature-inducible (BLT101) genes. Based on direct biochemical and indirect physiological evidence and similarity with a conserved protein domain in the Cluster of Orthologous Groups (COG) database (e.g., yeast PMP3), the rice genes belong to a class of low-molecular-weight hydrophobic proteins involved in maintaining the integrity of the plasma membrane during cold, dehydration and salt stress conditions. Both genes exhibit a genotype-specific expression signature characterized by early and late stress-inducible expression in tolerant and intolerant genotypes, respectively. The differences in temporal expression profiles are consistent with cultivar differences in cold-induced membrane leakiness and seedling vigor. The presence of CRT/DRE promoter cis-elements is consistent with the synchronized expression of OsLti6 genes with the C-repeat binding factor/drought responsive element-binding protein (CBF/DREB) transcriptional activator. The present results indicate that the Oslti6 genes are part of a battery of cold stress defense-related genes regulated by a common switch.