Functional Characterization Of Phytophthora Sojae CRN Effectors In Nicotiana Benthamiana

The hemibiotrophic oomycete pathogen, Phytophthora sojae, causes soybean stem and root rot. This destructive pathogen is a fungus-like microorganism and well known for the rapidity with which it overcomes host resistance. The microorganism employs diverse mechanisms to break down plant defences. Diverse type of transposons and many gene families are present in the genome which encodes the effector proteins involved in causing the pathogenicity. P. sojae with the genome of 95 Mb is predicted to secret hundreds of effector proteins inside the host plant cells to facilitate infection and colonization. The oomycete effector proteins are divided into two main classes, apoplastic effectors are secreted into the plant extracellular space, and cytoplasmic effectors are translocated inside the plant cell, where they target different subcellular compartments. The Crinklers (Crinkling and Necrosis, CRN), are cytoplasmic effectors that are conserved in oomycete pathogens and their encoding genes are highly expressed at the infectious stages in P. sojae. However, most of these effectors are functionally uncharacterized.In this study, we selected three effectors proteins PsCRN70, PsCRN161 and PsCRN115 to monitor their potential roles in planta and to develop a stable transformation procedure for investigating the defense responses in Nicotiana benthamiana plants. Above proteins belong to the CRN effectors protein family and contain conserved motif of LQLFLAK and HVLVVVP.We functionally characterized an effector gene PsCRN70 by transiently and stably overexpressing it in Nicotiana benthamiana. We demonstrated that PsCRN70 localized to the plant cell nucleus and suppressed cell death elicited by all the tested cell death-inducing proteins, including BAX, PsAvh241, PsCRN63, PsojNIP and R3a/Avr3a. Overexpression of the PsCRN70 gene in N. benthamiana enhanced susceptibility to P. parasitica. The H2O2 accumulations in the PsCRN70-transgenic plants were reduced compared to the GFP-lines. The transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX, were also down-regulated in the PsCRN70-transgenic lines. Our results suggest that PsCRN70 may function as a universal suppressor of the cell death induced by many elicitors, the host H2O2 accumulation and the transcription of the defense-associated genes, and therefore promote pathogen infection.PsCR.Nl61 of Phylophthora sojae is a member of the CRN class of cytoplasmic effectors with functions in avirulence, cell death suppression and tolerance to drought. Overexpression of PsCRN161 localized to the plant cell nucleus and enhanced the plant resistance to oomycete pathogens, Phytophthora parasitica or Phytophthora capsici by elevating the transcriptional levels of the defense-associated genes, including PR1b, PR2b, ERF1 and LOX. Importantly, PsCRN161-transgenic plants exhibited significantly higher H2O2 accumulations and shown upregulating expression of reactive oxygen species-related genes compared to GFP control plants. Moreover, many physiological analyses showed that the tolerance of PsCRN161-transgenic N. benthimiana increases towards salt and drought stresses. Overexpressed PsCRN161 gene in Nicotiana benthamiana showed suppressed cell death elicited by diverse elicitors including oomycete and bacteria pathogen effectors. The study confirmed that for the first time a direct evidence of countering drought stress tolerance in plant by genetic modification using a P. sojae gene. On the basis of our results we suggest that PsCRN161 is very important to spread infection, suppressor of cell death induced by many elicitors and the regulation of the salt and drought stress responses in plants. We generated independent transgenic Nicotiana benthamiana lines of PsCRN115 effector of Phytophthora sojae, and showed that PsCRN115 localized to the plant cell nucleus and enhanced the plant resistance to oomycete pathogens, Phytophthora parasitica or Phytophthora capsici by elevating the transcriptional levels of the defense-associated genes, including PRIb, PR2b, ERF1 and LOX. Importantly, PsCRN115-transgenic plants exhibited significantly higher H2O2 accumulations and shown upregulating expression of reactive oxygen species-related genes, including ROS-scavenging CAT, APX, SOD and GST and ROS-producing RbohA and RbohB compared to GFP control plants. Moreover, many physiological analyses showed that the tolerance of PsCRN115-transgenic N. benthimiana increases towards salt and drought stresses. Taken together, these results indicate that the PsCRN115 effector suppressed the infection of plant pathogens by manipulating the immune signalling pathway in plant cells.For better understanding of Phtophthora infection we have to understand different functional branches of the plant immune systems targeted by effector protein. On the basis of our results we suggest that through adaptation of genetic engineering techniques we can produce disease resistant crops.