The first three inoculated time points, 6, 12, and 24 hpi, had an average percentage of mapped reads between 85 and 88%. The last two time points, 36 and 48 hpi, where microscopic and macroscopic cell death was observed only had 52 and 64% of their reads mapped respectively. The complete results for total reads, percentage of reads mapped, and percentage of uniquely mapped reads are listed in Table 4.2.The total number of annotated proteins as well as DEGs are listed in Table 4.3. There were the highest number of both up-regulated and down-regulated DEGs at 24 hpi at 14,195 and 13,237 respectively. Using a cluster analysis of differentially expressed genes , the function of unknown genes can be recognized. In the hierarchical clustering, different areas with different colors, represent different groups of the cluster of genes up and down-regulated. This analysis shows two major clusters by treatment, one composed of all the mock uninoculated samples and inoculated samples corresponding to early time points . There were a large number of upregulated enriched genes associated with the ribosome and protein processing in the endoplasmic reticulum at 48-hpi, 321 and 189 respectively . There were also 72 enriched genes associated with the ribosome down-regulated at 12-hpi . Downregulated genes associated with the spliceosome were enriched at 12 and 36-hpi . There were many common differentially expressed plant defense genes that were upregulated in P. cinnamomi infected N. benthamiana leaves when compared to rootinoculated avocado, chestnut, and eucalyptus . Specifically, PAL, Thaumatin, Allene oxide synthase, F-box proteins, and cytochrome P450 were also significantly upregulated in avocado and L. longifolia roots. Genes encoding several members of the WRKY transcription factors were up-regulated in avocado, eucalyptus, chestnut, and L. longifolia roots. Glutathione S-transferase gene was up-regulated significantly in avocado and L. longifolia roots.
These and other defense related genes commonly expressed in avocado, round flower buckets other model systems, and the N. benthamiana pathosystem support the use of N. benthamiana to investigate defense gene response to P. cinnamomi.In this study we have elucidated the gene expression in response to P. cinnamomi infection in the N. benthamiana model system. By analyzing the transcriptome of N. benthamiana with RNAseq at five critical time points during the infection it was possible to identify important defense pathways using our model system. As early as 6-hpi there is a response by the infected host. We see a significant number of genes involved in the biosynthesis of secondary metabolites up-regulated. Specifically, known defense-related biosynthesis pathways such as flavonoid, terpenoid, and the phenilpropanoid pathways were enriched. Numerous plant-pathogen interaction genes were also enriched indicating the initiation of an active defense response to the pathogen. The highest number of enriched genes were involved in the biosynthesis of secondary metabolites at both 6 and 12-hpi. There was also a significant down-regulation of genes associated with the ribosome and plant hormone signal transduction especially auxin related genes. There is also a down-regulation in photosynthesis and metabolic pathways indicative of a decrease in resources allocated to growth and energy production in response to pathogen detection. The DEGs analysis confirms this early defense response with up-regulated hormone signaling, transcription factors, pathogen related genes, and resistance genes. At 24 hpi the KEGG enriched terms show a decrease in carbon fixation and metabolism which supports the allocation of resources towards plant defense. Salicylic acid binding is also down-regulated 5-fold which could be the result of pathogen effectors subverting the defense response. At the same time, we found transcription factors, PR genes, R genes, and anti-fungal genes that were up-regulated over 10-fold. Although P. cinnamomi is attempting to subvert the host defenses, the extensive colonization and intracellular growth at this stage of the infection has induced an extreme response in N. benthamiana.
At 36 hpi we found that besides significant up regulation in the plantpathogen interaction KEGG enrichment pathway there was up regulation of genes involved in endocytosis and phagosomes. Interestingly this is the time point where plant cell death becomes apparent in previously stained images of the infection process and is thought to be the stage where P. cinnamomi becomes necrotrophic in its infection strategy. There is also a 10-fold down regulation of SA binding and 5 to 10- fold up regulation of the JA pathway that further supports the necrotrophic infection occurring at this stage. At 48 hpi the KEGG pathway shows that 321 unigenes are involved in the up regulation of ribosome function. At the same time 770 unigenes are involved in the down regulation of metabolic pathways. Numerous defense genes are also being highly expressed at this time point including the continued up-regulation of the JA pathway. PAMP-triggered immunity is the plants first layer of defense against plant pathogens. Plants have developed pattern recognition receptors that initiate a defense response before the pathogen is able to infect the plant. This early PTI response is at a time in our model system when many of the encysted zoospores on the inoculated leaf surface haven’t germinated and there is no intracellular penetration . PTI response is linked to reactive oxygen species production, lignin and callose reinforcement, and the up-regulation of pathogenesis-related genes. Genes encoding Glutathione S-transferase, which serves to protect plant cells from ROS production was significantly up-regulated in our system as well as previously identified as being up-regulated during infection in Arabidopsis, Z. mays, and avocado . PR-genes up-regulated significantly in N. benthamiana in response to P. cinnamomi infection included; PR-1, PR-4 , PR-5 , and PR-9 . PR-5 was also up-regulated in infected avocado roots and PR-1 and PR-5 were up-regulated in eucalyptus roots . Hormone signaling plays an important role in a plants response to various pathogens and the pathway initiated varies depending on the type of pathogen. The jasmonic acid and ethylene signaling pathways are normally initiated in response to necrotrophic pathogens.
The cytochrome P450 super family is the largest enzymatic protein family in plants . CYP genes are involved in hormone signaling and associated with the JA pathway . CYP has been described as a JA-responsive gene which was up-regulated 37.75-fold in L. longifolia . Numerous genes in this family were up-regulated in all of the five time points we analyzed. Up-regulation of CYP has also been found in inoculated avocado . Some interesting and less well described anti-fungal genes in the CYP family, premnaspirodiene oxygenase and aristolochene synthase were identified that would be good candidates for further analysis. F-box proteins associated with the JA pathway were upregulated in 4 out of 5 of the time points in our system. The up-regulation of this gene in response to P. cinnamomi infection has also been discovered in avocado as well as numerous other model systems indicating a similar response among these different plants. Allene oxide synthase genes involved in JA biosynthesis , were also up-regulated in our system. JA response is traditionally associated with necrotrophic pathogen defense, plastic flower buckets wholesale but recent studies have shown that biotrophic pathogens such as Plasmopara viticola and hemi-biotrophs like P. infestans and P. cinnamomi can also trigger the activation of a JA triggered response. There were also some differentially expressed genes that are normally associated with the SA pathway discovered in the N. benthamiana system including up-regulated PAL genes and down-regulated SA binding genes. PAL is the key enzyme for the phenylpropanoid pathway which is involved in SA biosynthesis, lignin, and antimicrobial compounds such as flavonoids and phytoalexins . Auxin signaling has been shown to play and important role in the induction of resistance to P. cinnamomi . Plants using more than one defense pathway in response to P. cinnamomi infection has been seen previously in avocado and L. longifolia but was not found in Z. mays . Interestingly, genes encoding WRKY transcription factors were significantly upregulated at all of the time points in our system. In plants, WRKY transcription factors are encoded by a large family of genes, and they are involved in abiotic and biotic stress and are activated by pathogen perception . WRKY genes have been previously shown to be direct transcriptional targets of NPR1 in response to SA abundance . The significant up-regulation of WRKY transcription factors in our model system may indicate that there is some JA/ SA crosstalk in the infected samples. Up regulation of WRKY transcription factors in response to P. cinnamomi infection was also found in eucalyptus, chestnut, and L. longifolia roots. . WRKY51 transcription factor was selected as an ideal candidate for functional experiments because of its consistent up-regulated expression from 12 to 48 hpi shown in the RNAseq data and the uniform results during the qPCR validation. For the many reasons listed above WRKY51 transcription factor was chosen to functionally validate in our N. benthamiana model system. The transient over-expression of the WRKY51 construct was carefully timed through numerous experiments to discover at what point, if any, in comparison with the P. cinnamomi infection would the expression of this transcription factor have the greatest effect on the colonization of the pathogen. Transient expression of the WRKY51 protein 3 hours before inoculation with P. cinnamomi zoospores produced the clearest phenotype difference between the experimental and control groups. This early transient expression may induce an early defense response before the pathogen has a chance to colonize the host.
The RNAseq data in this study and others, and the functional assay using WRKY51 confirm that a close analysis of its expression in avocado is warranted. The RNAseq data as well as the functional work in this study provide a wealth of information concerning host defense response to P. cinnamomi infection. It is important however, to begin to make connections back to avocado which is the economically important crop we are interested in understanding more completely. By using the data obtained in this study and developing functional experiments that overcome the limitations associated with tree crops such as avocado we will be able to better address the long-term breeding concerns of avocado growers. Genes identified in our model system such as cytochrome P450 or identified and validated such as WRKY51 canbe used for marker assisted breeding. The avocado qPCR data for cytochrome P450 is an important first step in the goal to learn more about avocado defense gene response directly. The expression of cytochrome P450 in inoculated detached avocado leaves was similar to the expression in detached N. benthamiana leaves in both PS.54 and Dusa® rootstocks. Since cytochrome P450 has already been identified to be significantly up-regulated in P. cinnamomi infected avocado roots , it is reasonable to infer that this defense gene is similarly expressed in both roots and shoots. These universally expressed plant defense genes will provide vital information for resistance breeding projects in avocado. The next step is to find a functionally validated defense gene in our model system that is differentially expressed in the susceptible and tolerant avocado varieties and also transiently over express the avocado candidate genes homologs in N. benthamiana for functional validation. This would be an ideal candidate for marker assisted breeding. Using our model system, it is possible to identify such a candidate.Plant-parasitic nematodes infect a broad range of commercially important crop families such as the Solanaceae , Fabaceae , Malvaceae , Amaranthaceae , and Poaceae , causing an estimated annual loss of $80 billion USD . The most economically important group of PPNs are sedentary endoparasites, including root-knot nematodes and cyst nematodes . Sedentary endoparasites induce the formation of permanent feeding cells that provide specialized nutrient sources for nematodes . Infective second-stage juveniles of RKNs predominantly invade near the root tip and then migrate intercellularly toward the apical meristematic region without crossing the endodermis, making a U-turn to enter the vascular cylinder where they induce several giant cells as a feeding site by stimulating the redifferentiation of root cells into multi-nucleate giant cells by repeated nuclear divisions without cytoplasmic division. After maturation, adult RKN females lay eggs in a gelatinous egg mass on or below the surface of the root . In contrast, CNs move destructively through cells into the vascular cylinder, select a single cell, and form a syncytium as a feeding site by local dissolution of cell walls and protoplast fusion of neighboring cells. A CN female produces hundreds of eggs and its body forms a cyst that can protect the eggs for many years in the soil .