Comparative proteomics combined with analyses of transgenic plants reveal ZmREM1.3 mediates maize resistance to southern corn rust

Southern corn rust (SCR), which is a destructive disease caused by Puccinia polysora Underw. (P. polysora), commonly occurs in warm‐temperate and tropical regions. To identify candidate proteins related to SCR resistance and characterize the molecular mechanisms underlying the maize–P. polysora interaction, a comparative proteomic analysis of susceptible and resistant maize lines was performed. Statistical analyses revealed 1489 differentially abundant proteins in the resistant line, as well as 1035 differentially abundant proteins in the susceptible line. After the P. polysora infection, the abundance of one remorin protein (ZmREM1.3) increased in the resistant genotype, but decreased in the susceptible genotype. Plant‐specific remorins are important for responses to microbial infections as well as plant signalling processes. In this study, transgenic maize plants overexpressing ZmREM1.3 exhibited enhanced resistance to the biotrophic P. polysora. In contrast, homozygous ZmREM1.3 UniformMu mutant plants were significantly more susceptible to P. polysora than wild‐type plants. Additionally, the ZmREM1.3‐overexpressing plants accumulated more salicylic acid (SA) and jasmonic acid (JA). Moreover, the expression levels of defence‐related genes were higher in ZmREM1.3‐overexpressing maize plants than in non‐transgenic control plants in response to the P. polysora infection. Overall, our results provide evidence that ZmREM1.3 positively regulates maize defences against P. polysora likely via SA/JA‐mediated defence signalling pathways. This study represents the first large‐scale proteomic analysis of the molecular mechanisms underlying the maize–P. polysora interaction. This is also the first report confirming the remorin protein family affects plant resistance to SCR.     

Expression of tomato salicylic acid (SA)‐responsive pathogenesis‐related genes in Mi‐1‐mediated and SA‐induced resistance to root‐knot nematodes

The expression pattern of pathogenesis‐related genes PR‐1, PR‐2 and PR‐5, considered as markers for salicylic acid (SA)‐dependent systemic acquired resistance (SAR), was examined in the roots and shoots of tomato plants pre‐treated with SA and subsequently infected with root‐knot nematodes (RKNs) (Meloidogyne incognita). PR‐1 was up‐regulated in both roots and shoots of SA‐treated plants, whereas the expression of PR‐5 was enhanced only in roots. The over‐expression of PR‐1 in the whole plant occurred as soon as 1 day after SA treatment. Up‐regulation of the PR‐1 gene was considered to be the main marker of SAR elicitation. One day after treatment, plants were inoculated with active juveniles (J2s) of M. incognita. The number of J2s that entered the roots and started to develop was significantly lower in SA‐treated than in untreated plants at 5 and 15 days after inoculation. The expression pattern of PR‐1, PR‐2 and PR‐5 was also examined in the roots and shoots of susceptible and Mi‐1‐carrying resistant tomato plants infected by RKNs. Nematode infection produced a down‐regulation of PR genes in both roots and shoots of SA‐treated and untreated plants, and in roots of Mi‐carrying resistant plants. Moreover, in resistant infected plants, PR gene expression, in particular PR‐1 gene expression, was highly induced in shoots. Thus, nematode infection was demonstrated to elicit SAR in shoots of resistant plants. The data presented in this study show that the repression of host defence SA signalling is associated with the successful development of RKNs, and that SA exogenously added as a soil drench is able to trigger a SAR‐like response to RKNs in tomato.     

Overexpression of AtPAD4 in transgenic Brachypodium distachyon enhances resistance to Puccinia brachypodii

• Brachypodium distachyon (L.) has recently emerged as a model for temperate grasses for investigating the molecular basis of plant–pathogen interactions. Phytoalexin deficient 4 (PAD4) plays a regulatory role in mediating expression of genes involved in plant defence.
• In this research, we generated transgenic B. distachyon plants constitutively overexpressing AtPAD4. Two transgenic B. distachyon lines were verified using PCR and GUS phenotype.
• Constitutive expression of AtPAD4 in B. distachyon enhanced resistance to Puccinia brachypodii. P. brachypodii generated less urediniospores on transgenic than on wild‐type plants. AtPAD4 overexpression enhanced salicylic acid (SA) levels in B. distachyon‐infected tissues. qRT‐PCR showed that expression of pathogenesis‐related 1 (PR1) and other defence‐related genes were up‐regulated in transformed B. distachyon following infection with P. brachypodii.
• Our results indicate that AtPAD4 overexpression in B. distachyon plants led to SA accumulation and induced PR gene expression that reduced the rate of colonisation by P. brachypodii.

     

Transcriptome Analysis Identifies Plasmodiophora brassicae Secondary Infection Effector Candidates

Plasmodiophora brassicae (Wor.) is an obligate intracellular plant pathogen affecting Brassicas worldwide. Identification of effector proteins is key to understanding the interaction between P. brassicae and its susceptible host plants. To date, there is very little information available on putative effector proteins secreted by P. brassicae during a secondary infection of susceptible host plants, resulting in root gall production. A bioinformatics pipeline approach to RNA‐Seq data from Arabidopsis thaliana (L.) Heynh. root tissues at 17, 20, and 24 d postinoculation (dpi) identified 32 small secreted P. brassicae proteins (SSPbPs) that were highly expressed over this secondary infection time frame. Functional signal peptides were confirmed for 31 of the SSPbPs, supporting the accuracy of the pipeline designed to identify secreted proteins. Expression profiles at 0, 2, 5, 7, 14, 21, and 28 dpi verified the involvement of some of the SSPbPs in secondary infection. For seven of the SSPbPs, a functional domain was identified using Blast2GO and 3D structure analysis and domain functionality was confirmed for SSPbP22, a kinase localized to the cytoplasm and nucleus.     

The impact of Pseudomonas syringae type III effectors on transient protein expression in tobacco

The production of recombinant proteins in plants is often achieved by transient expression, e.g. following the injection or vacuum infiltration of Agrobacterium tumefaciens into tobacco leaves. We investigated the associated plant defence responses, revealing that callose deposition is triggered by T–DNA transfer and that subsets of secondary metabolites accumulate in response to mechanical wounding or the presence of bacteria. We also tested the ability of five co‐expressed type III effector proteins from Pseudomonas syringae to modulate these defence responses and increase the yield of two model proteins, the fluorescent marker DsRed and monoclonal antibody 2G12. HopF2 and AvrRpt2 induced necrotic lesions 5 days post‐injection (dpi) even at low doses (OD_600 nm = 0.0078), and increased the concentration of certain secondary metabolites. HopAO1 significantly reduced the number of callose deposits at 2 dpi compared to cells expressing DsRed and 2G12 alone, whereas HopI1 reduced the concentration of several secondary metabolites at 5 dpi compared to cells expressing DsRed and 2G12 alone. Co‐expression with HopAO1, AvrPtoB or HopI1 increased the concentrations of DsRed and 2G12 increased by ~6% but this was not a significant change. In contrast, HopF2 and AvrRpt2 significantly reduced the concentrations of DsRed and 2G12 by 34% and 22%, respectively. Our results show that type III effector proteins can modulate plant defence responses and secondary metabolite profiles but that transient co‐expression is not sufficient to increase the yields of target recombinant proteins in tobacco.     

Mechanisms of resistance in the rice cultivar Manikpukha to the rice stem nematode Ditylenchus angustus

The incompatible interaction between the rice cultivar Manikpukha and the rice stem nematode Ditylenchus angustus has been reported recently. This research focuses on the underlying mechanisms of resistance in Manikpukha. Invasion, post‐infection development and reproduction of D. angustus were compared in compatible and incompatible interactions to identify the stage in which resistance occurs. The results indicate that resistance in Manikpukha is associated with reduced development and reproduction, implying that resistance acts post‐invasion. We studied the possible involvement of three classical defence hormones, salicylic acid (SA), jasmonic acid (JA) and ethylene (ET), in response to infection in a compatible interaction using biosynthesis/signalling‐deficient transgenic rice lines. All three hormones appear to have an influence on the basal defence of Nipponbare against the stem nematode. Although hormone application increases basal defences, expression studies and hormone analyses after nematode infection in Manikpukha did not show a clear involvement of the hormone defense pathways for SA, ET and JA. However, it seems that OsPAL1 plays a pivotal role in resistance, indicating that the phenylpropanoid pathway and its products might be key players in the incompatible interaction. Lignin measurement showed that, although basal levels are similar, Manikpukha had a significantly higher lignin content on nematode infection, whereas it was decreased in the susceptible cultivar. The results presented here show that SA, ET and JA are involved in basal defences, but the resistance of Manikpukha against D. angustus probably relies on products of the phenylpropanoid pathway.