Zhaohui Chu

Rutin-Mediated Priming of Plant Resistance to Three Bacterial Pathogens Initiating the Early SA Signal Pathway

Flavonoids are ubiquitous in the plant kingdom and have many diverse functions, including UV protection, auxin transport inhibition, allelopathy, flower coloring and insect resistance. Here we show that rutin, a proud member of the flavonoid family, could be functional as an activator to improve plant disease resistances. Three plant species pretreated with 2 mM rutin were found to enhance resistance to Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Pseudomonas syringae pv. tomato strain DC3000 in rice, tobacco and Arabidopsis thaliana respectively. While they were normally propagated on the cultural medium supplemented with 2 mM rutin for those pathogenic bacteria. The enhanced resistance was associated with primed expression of several pathogenesis-related genes. We also demonstrated that the rutin-mediated priming resistance was attenuated in npr1, eds1, eds5, pad4-1, ndr1 mutants, and NahG transgenic Arabidopsis plant, while not in either snc1-11, ein2-5 or jar1 mutants. We concluded that the rutin-priming defense signal was modulated by the salicylic acid (SA)-dependent pathway from an early stage upstream of NDR1 and EDS1.

Domain dissection of AvrRxo1 for suppressor, avirulence and cytotoxicity functions.

AvrRxo1, a type III effector from Xanthomonas oryzae pv. oryzicola (Xoc) which causes bacterial leaf streak (BLS) in rice, can be recognised by non-host resistance protein Rxo1. It triggers a hypersensitive response (HR) in maize. Little is known regarding the virulence function of AvrRxo1. In this study, we determined that AvrRxo1 is able to suppress the HR caused by the non-host resistance recognition of Xanthomonas oryzae pv. oryzae (Xoo) by Nicotiana benthamiana. It is toxic, inducing cell death from transient expression in N. benthamiana, as well as in yeast. Among the four AvrRxo1 alleles from different Xoc strains, we concluded that the toxicity is abolished by a single amino acid substitution at residue 344 in two AvrRxo1 alleles. A series of truncations from the carboxyl terminus (C-terminus) indicate that the complete C-terminus of AvrRxo1 plays an essential role as a suppressor or cytotoxic protein. The C-terminus was also required for the avirulence function, but the last two residues were not necessary. The first 52 amino acids of N-terminus are unessential for toxicity. Point mutagenesis experiments indicate that the ATP/GTP binding site motif A is required for all three functions of AvrRxo1, and NLS is required for both the avirulence and the suppression of non-host resistance. The putative thiol protease site is only required for the cytotoxicity function. These results determine that AvrRxo1 plays a role in the complex interaction with host proteins after delivery into plant cells.

Development of Marker-Free Transgenic Potato Tubers Enriched in Caffeoylquinic Acids and Flavonols

Potato (Solanum tuberosum L.) is a major crop worldwide that meets human economic and nutritional requirements. Potato has several advantages over other crops: easy to cultivate and store, cheap to consume, and rich in a variety of secondary metabolites. In this study, we generated three marker-free transgenic potato lines that expressed the Arabidopsis thaliana flavonol-specific transcriptional activator AtMYB12 driven by the tuber-specific promoter Patatin. Marker-free potato tubers displayed increased amounts of caffeoylquinic acids (CQAs) (3.35-fold increases on average) and flavonols (4.50-fold increase on average). Concentrations of these metabolites were associated with the enhanced expression of genes in the CQA and flavonol biosynthesis pathways. Accumulation of CQAs and flavonols resulted in 2-fold higher antioxidant capacity compared to wild-type potatoes. Tubers from these marker-free transgenic potatoes have therefore improved antioxidant properties.

Overexpression of OsDT11 , which encodes a novel cysteine-rich peptide, enhances drought tolerance and increases ABA concentration in rice

Short-chain peptides play important roles in plant development and responses to abiotic and biotic stresses. Here, we characterized a gene of unknown function termed OsDT11, which encodes an 88 amino acid short-chain peptide and belongs to the cysteine-rich peptide family. It was found that the expression of OsDT11 can be activated by polyethylene glycol (PEG) treatment. Compared with wild-type lines, the OsDT11-overexpression lines displayed dramatically enhanced tolerance to drought and had reduced water loss, reduced stomatal density, and an increased the concentration of abscisic acid (ABA). The suppression of OsDT11 expression resulted in an increased sensitivity to drought compared to wild-type expression. Several drought-related genes, including genes encoding abscisic acid (ABA) signaling markers, were also strongly induced in the OsDT11-overexpressing lines. Moreover, the expression of OsDT11 was repressed in ABA-insensitive mutant Osbzip23 and Os2H16 RNAi lines. These results suggest that OsDT11-mediated drought tolerance may be dependent on the ABA signaling pathway.

Identification of two novel Rhizoctonia solani -inducible cis -acting elements in the promoter of the maize gene, GRMZM2G315431

Plants are continuously exposed to myriad pathogen stresses. However, the molecular mechanisms by which these stress signals are perceived and transduced are poorly understood. In this study, the maize gene GRMZM2G315431 was identified to be highly inducible by Rhizoctonia solani infection, suggesting that the promoter of GRMZM2G315431 (pGRMZM2G315431) might contain a specific cis-acting element responsive to R. solani attack. To identify the R. solani-responsive element in pGRMZM2G315431, a series of binary plant transformation vectors were constructed by fusing pGRMZM2G315431 or its deletion-derivatives with the reporter genes. In the transient gene expression system of Nicotiana benthamiana leaves inoculated with R. solani, GUS quantification suggested that the DNA fragment contains the unknown pathogen-inducible cis-elements in the −1323 to −1212 region. Furthermore, detailed quantitative assays showed that two novel cis-elements, GTTGA in the −1243 to −1239 region and TATTT in the −1232 to −1228 region, were responsible for the R. solani-inducible activity. These two cis-elements were also identified to have R. solani-specific-inducible activity in stable transgenic rice plants, suggesting the existence of a novel regulation mechanism involved in the interaction between R. solani and Zea mays.

Identification of Anthocyanin Composition and Functional Analysis of an Anthocyanin Activator in Solanum nigrum Fruits

Solanum nigrum fruits have been conventionally used in beverages due to their nutritional substances such as minerals, vitamins, amino acids, proteins, sugars, polyphenols, and anthocyanins. The characterization of components and regulatory mechanism of anthocyanins in S. nigrum fruits have rarely been reported. In this study, we determined that the peel and flesh of S. nigrum fruits shared similar HPLC profiles but different contents and total antioxidant activities for anthocyanins. After an efficient purification method, mainly including extraction with pH 1.0 distilled water and then desorption with pH 1.0 95% ethanol after a DM-130 resin adsorption step to obtain more pure anthocyanin extracts, the purity of anthocyanins extracted from S. nigrum fruits reached 56.1%. Moreover, eight anthocyanins from S. nigrum fruit were identified with HPLC-MS/MS for the first time. A typical R2R3-MYB transcription factor gene, SnMYB, was also cloned for the first time by rapid amplification of cDNA ends (RACE)-PCR from S. nigrum. Moreover, the contents of anthocyanins were shown to correlate well (r = 0.93) with the expression levels of SnMYB gene during the fruit’s developmental stages. Most significantly, SnMYB gene successfully produced high anthocyanin content (1.03 mg/g) when SnMYB gene was transiently expressed in tobacco leaves. Taken together, S. nigrum fruits are a promising resource for anthocyanin extraction, and SnMYB gene is an activator that positively regulates anthocyanin biosynthesis in S. nigrum.

The polygalacturonase-inhibiting protein 4 (OsPGIP4), a potential component of the qBlsr5a locus, confers resistance to bacterial leaf streak in rice.

AbstractMain conclusionOsPGIP4overexpression enhances resistance to bacterial leaf streak in rice. Polygalacturonase-inhibiting proteins are thought to play important roles in the innate immunity of rice against fungi. Here, we show that the chromosomal location of OsPGIP4 coincides with the major bacterial leaf streak resistance quantitative trait locus qBlsr5a on the short arm of chromosome 5. OsPGIP4 expression was up-regulated upon inoculation with the pathogen Xanthomonas oryzae pv. oryzicola strain RS105. OsPGIP4 overexpression enhanced the resistance of the susceptible rice variety Zhonghua 11 to RS105. In contrast, repressing OsPGIP4 expression resulted in an increase in disease lesions caused by RS105 in Zhonghua 11 and in Acc8558, a qBlsr5a resistance donor. More interestingly, upon inoculation, the activated expression of pathogenesis-related genes was attenuated for those genes involved in the salicylic acid pathway, while the activated expression of jasmonic acid pathway markers was increased in the overexpression lines. Our results not only provide the first report that rice PGIP could enhance resistant against a bacterial pathogen but also indicate that OsPGIP4 is a potential component of the qBlsr5a locus for bacterial leaf streak in rice.

OsASR2 regulates the expression of a defence‐related gene, Os2H16, by targeting the GT‐1 cis‐element

Summary The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.

Improvement of Verticillium Wilt Resistance by Applying Arbuscular Mycorrhizal Fungi to a Cotton Variety with High Symbiotic Efficiency under Field Conditions

Arbuscular mycorrhizal fungi (AMF) play an important role in nutrient cycling processes and plant stress resistance. To evaluate the effect of Rhizophagus irregularis CD1 on plant growth promotion (PGP) and Verticillium wilt disease, the symbiotic efficiency of AMF (SEA) was first investigated over a range of 3% to 94% in 17 cotton varieties. The high-SEA subgroup had significant PGP effects in a greenhouse. From these results, the highest-SEA variety of Lumian 1 was selected for a two-year field assay. Consistent with the performance from the greenhouse, the AMF-mediated PGP of Lumian 1 also produced significant results, including an increased plant height, stem diameter, number of petioles, and phosphorus content. Compared with the mock treatment, AMF colonization obviously inhibited the symptom development of Verticillium dahliae and more strongly elevated the expression of pathogenesis-related genes and lignin synthesis-related genes. These results suggest that AMF colonization could lead to the mycorrhiza-induced resistance (MIR) of Lumian 1 to V. dahliae. Interestingly, our results indicated that the AMF endosymbiont could directly inhibit the growth of phytopathogenic fungi including V. dahliae by releasing undefined volatiles. In summary, our results suggest that stronger effects of AMF application result from the high-SEA.

Functional analysis of the GRMZM2G174449 promoter to identify Rhizoctonia solani -inducible cis -elements in maize

BackgroundBanded leaf and sheath blight (BLSB), caused by the necrotrophic fungus Rhizoctonia solani, is a highly devastating disease in most maize and rice growing areas of the world. However, the molecular mechanisms of perceiving pathogen signals are poorly understood in hosts.ResultsHere, we identified a Rhizoctonia solani-inducible promoter pGRMZM2G174449 in maize. Deletion analysis showed that the −574 to −455 fragment was necessary for pGRMZM2G174449 in responding to R. solani and this fragment contained the unknown pathogen-inducible cis-elements according to the bioinformatics analysis. Furthermore, detailed quantitative assays showed that two cis-elements, GCTGA in the −567 to −563 region and TATAT in the −485 to −481 region, were specifically responsible for the R. solani-inducible activity. A series of point mutation analysis indicated that the two cis-elements have the conserved motifs of NHWGN and DWYWT, respectively.ConclusionOur results indicated that pGRMZM2G174449 is a good R. solani-inducible promoter suitable for genetic engineering of BLSB resistance. And NHWGN and DWYWT are two R. solani-inducible cis-elements that play important roles in pGRMZM2G174449 responding to R. solani.