Introduction
Several diseases in plants can be caused due to attack and infection by some bacteria species. Preliminary studies have shown that usually bacteria and plants have chemical communication. Some bacteria are residents while others are transient. However, the infection of plants may be beneficial or harmful. Research on plant-associated bacteria has extensively been studied during the last years and mechanisms of bacterial pathogenicity are becoming well-known. Understanding the mechanism of symbiosis between them should also provide a relevant breakthrough for the defence mechanism of plants. However, pathogenomics and more specifically how plants interact with microorganisms remain challengeable. In fact, a series of techniques are already available to identify genes related to adaptation of bacteria in plants and determine the role of their gene products in bacteria-plant interactions in vivo. Examples of these techniques are the IVET (In Vitro Expression Technology), RIVET (Reverse IVET), STM (Signature-tagged mutagenesis), DFI (Difference fluorescence induction), expression microarrays and RNA seq. All of the above techniques have been used successfully and have determined novel factors involving in pathogen adaptations. However, they have some limitations compared to the new suggested screening system from Fikowicz-Krosko and Czajkowski (2017), including the requirement of specialised laboratory equipment, advanced technical assistance and some of them can produce economic issues (Handfield and Levesque, 1999).This is crucial for several fields such as agriculture, biotechnology, economy or food industry.
One of the top-ten plant pathogenic bacteria species in molecular plant pathology is Dickeya spp (previously named pectinolytic Erwinia spp.) which infect a number of plants species including important crops, such as potato Solanum tuberosum, resulting to economic losses in seed potato production worldwide (Toth and Birch, 2005; Mansfield et al., 2012) . The impact of Dickeya spp in potato epidemiology is huge. In particular, a pectinolytic plant-pathogenic bacterium, namely Dickeya solani, has been isolated from diseased potato and intrigues the interest since then for further studies (Sławiak et al., 2009; van der Wolf et al., 2014). Its significance in the epidemiology of potato has been reported due to the exhibition of blackleg like conditions in field-grown plants, the slow wilt symptoms, causing a soft rot of tubers. As a result, it affects potato production in a number of European countries plus Israel and Georgia (Toth et al., 2011). This phenomenon has been observed growing over the past ten years and has been confirmed by research in different European countries, such as Netherlands (Czajkowski, Grabe and van der Wolf, 2009), Finland (Laurila et al., 2008), and Poland (Lojkowska E, 2010). Thus, Dickeya spp are considered to be critical bacterial pathogens particularly in Europe and other countries due to its fast spreading.
Recent studies have shown that D. solani is active in a wide range of temperature and also capable of surviving in surface water for a long time (Laurila et al., 2008; Czajkowski et al., 2017). Moreover, it can infect plants in a range of natural conditions. Nevertheless, depth knowledge of the ecology of D. solani is not sufficient in order to demystify regulation of infection-related gene expression especially in the early stages of adaptation resulting to the colonization of plant tissues.
Experimental approach
Fikowicz-Krosko and Czajkowski (2017), have reported a new screening method in order to preselect D. solani Tn5 mutants which contain an inserted reporter gene in plant tissue-induced genes. The aim is to use these candidates directly for follow-up studies about the identity of the plant-induced genes and the role of their gene products in bacteria-plant interactions. Firstly, they have applied reporter gene technology which is widely used to monitor cellular events such as gene transfer and gene expression. This technology has the ability to “reports” effects of signalling events on gene expression inside cells. Advantages that can be obtained by this technology is their high sensitivity, reliability, convenience, and adaptability to large-scale measurements (Naylor, 1999). It is generally accepted as a biological screen in detection methods. In this review, an inducible promoterless gene, gusA, which encodes ß-glucuronidase, has been inserted in bacterium genome through random mutagenesis. In particular, mini-Tn5 transposon carrying gusA gene has been transformed in bacterium genome, generating Tn5 mutants. Transposon mutagenesis is widely used to insert single copies of a mini-transposon into the genome and disrupt genes leading to phenotypic changes. Achieve of the technique is to deliver exogenous genes, reporter genes, and then the expression of the reporter genes can help to determine essential genes in other processes. In this case, they are looking for genes involving in bacteria-plant interactions. In addition, mini-transposons include genes that provide resistance to antibiotics as selection markers. These preparations will provide the materials to screen for ß-glucuronidase positive phenotypes in the presence of plant tissues and X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) as a substrate of the enzyme.
The experimental procedure designed by the authors leads to a fast and reliable screening method with a upper goal the identification of up-regulated genes in early stages of bacterial adaptations with plants. At first, D. solani IPO2222 Tn5 mutants have been generated by random transposon mutagenesis. The Tn5 transposon has been donor from Escherichia coli (S17 λ-pir pFaj1819) cells. Following, ß-glucuronidase negative phenotypes have been selected under non-inductive conditions. The gusA gene is promoterless and cannot be expressed only if transposon jumps into the bacterium genome where a promoter can induce it. Therefore, when the transposon jumps downstream of a promoter, is expressed (positive colonies) or not expressed (negative colonies). For that reason, negative colonies have been selected. This makes sure that expression of ß-glucuronidase is only induced and monitored by plant tissues in the next-step experiments. After a 2-day incubation at 48 ºC of Tn5 mutants and plant tissues mixture, ß-glucuronidase positive phenotypes coloured blue and have been selected for further analysis. The blue colour is presented because of X-gal degradation by the ß-glucuronidase enzyme. At the end of the experiments, positive reaction with blue colour in the presence of plant tissues represent up-regulation of Tn5-disrupted gene in the bacterium genome. Therefore, Tn5 mutants with positive phenotypes can be collected for sequencing and further analysis to monitor their contribution in bacteria-plant interactions.
The plant tissues that have been used were leaves, stems and roots from Solanum tuberosum, a potato species, as a primary host and Solanum ducamara, a bittersweet nightshade, as a secondary host. The choice of plants was relevant knowing that this specific bacterium can cause damages to these economically important crops. Thus, identification of genes that contribute to bacteria adaptation will help in agriculture and also in the field of biotechnology to prevent or reduce this phenomenon. Moreover, for positive control, they have been used Tn5 mutants with ß-glucuronidase positive phenotypes in the presence of plant tissues. Negative controls have been used per mutant, containing only growth medium and Tn5 mutant without any type of plant tissue. Proper controls help to revise the results and confirm the reliability of the experiments. Also, it will exclude the possibility of the gene expression by other factors instead of plant tissues. In this case, negative control without plant tissues illustrates no expression of the ß-glucuronidase in all mutant sample. Thus, any blue colour refers to up-regulation of genes by plant tissues. At the same time, positive control can provide the intensity of the optimal colour when the enzyme is expressed. The intensity can probably associate with how strongly the plant tissue monitors the gene expression. Hence, the appropriate controls have been used in order to work out their results.
Description and analysis of data
At first, the group conjugate E. coli cells, carrying a suicide plasmid with the mini-transposon, with D. solani IPO2222 strain. They plated out samples on agar medium under non-inductive conditions and they selected negative colonies where ß-glucuronidase is not expressed. Nevertheless, the way of distinction between positive and negative colonies is not clear in the paper. There is no mention of the selective marker or any other technique that is been used to detect expression in particular colonies. Take into consideration that gusA gene is promoterless, negative phenotypes have been chosen to ensure that expression of the enzyme only controlled by the plant tissues. In the next step, negative phenotypes were incubated for 2-days at 48 ºC in the presence of plant tissues like root, stem and leaf and also X-gal, as a substrate of ß-glucuronidase. The temperature of incubation has not been supported, contrariwise recently previous studies have found that at 37 ºC majority of up-regulated genes have been induced (Czajkowski et al., 2017). Another point is the concentration of bacteria needed for plant infection. In normal conditions, a higher population of bacteria, about 106 CFU (colony forming units) is required and here they use 109, a sufficient concentration to have successful results.
Not all but some examples of the data are shown in the article. This cannot give us all the picture of the experiments. For example, experiment has been applied in two different plant species. According to the figure of the paper, only in one of the species particularly in potato and more specifically in the presence of leaf and stem tissue does enzyme expression appear. Indeed, it is well-known from the bibliography that Dickeya species infect potato tissues and therefore results are somehow expected. But, this does not mean that in the other species this will not happen. Although the point of this work is to illustrate a fast screening method, reliability of the system should be more evident.
According to the Figure 1, some interesting results have been observed for Tn5 mutants 2,3 and 4. Mutant 2 and 4 indicate positive phenotype of ß-glucuronidase in the presence of S. tuberosum stem tissue whereas mutant 3 has negative phenotype but positive phenotype in the presence of S. tuberosum leaf tissue. These phenotypes lead to the conclusion that expression of the enzyme probably is monitored by these particular plant tissues. Therefore, up-regulated genes are induced by plant tissues and should be selected for sequencing as previously have done (Czajkowski et al., 2017).
Figure 1: Presentation of example results of the high-throughput screening system to preselect plant tissue-induced D. solani Tn5 mutants.
(a) Magnification of wells containing Tn5 mutant cultures in the presence of several plant tissues. (b) Zoom in of M2 and M4 mutants showing a ß-glucuronidase positive phenotype in the presence of S. tuberosum stem tissue whereas M3 mutant has a positive phenotype in the presence of S. tuberosum leaf tissue. (c) Presentation of wells containing D. solani Tn5 WN2 mutant with ß-glucuronidase positive phenotype independent of the presence of plant tissue. (Adapted from Fikowicz-Krosko and Czajkowski, (2017) )
Data from the experiments are presented only in one clear and good quality figure. As regards to the quality of the data, it is obvious that this screening system is work but the good efficacy of it is not supported. For instance, mutants with positive phenotypes indicate light blue colour, as it can be seen. This raises an interesting question, where if this is due to how strong the induction from the promoter is to induce gene expression or if the method has limitations. Limitations can be the fact that something else induces the expression instead of plant tissue and this can only be confirmed with follow-up experiments. In addition, Fikowicz-Krosko and Czajkowski, point out that from the 500 D. solani IPO2222 negative colonies they collected from the beginning, 2.4 %, 10.4% and 4.4% of them had a ß-glucuronidase positive phenotype in contact with roots, stems, leaves of S. tuberosum respectively. As a result, stem tissues up-regulate genes more often compare to the other tissues. Any substantial evidence of this conclusions is not illustrated in their experiments. However, again the main idea of the article is to introduce a fast screening method but to confirm its efficiency further analysis of the samples is needed. This leads to the conclusion that the article suggests a screening method which seems to be easy and quick but there is no evidence that the extracted results are reliable.
Justification of the results and further improvements
The article is presenting a screening system that it is believed to be faster than previous genetic techniques and also reliable. The upper goal is to design an inexpensive and efficient method which allows preselection of candidate mutants and use of them directly for further studies. Therefore, they suggest that this method will be one of the first steps for further analysation of host and non-host plant interactions with bacteria and also with other biotic and abiotic stressors. Moreover, it can earn time during laboratory work. However, they underline the fact that this system is only the preliminary work and in order to assess the gene expression, a quantitative manner is needed. Without a doubt is an interesting approach and will release many hands in laboratory considering that no equipment is required.
Authors mentioned that they also propose this method in order to evaluate the interactions between D. solani IPO2222 with bittersweet nightshade S. dulcamara and potato S. tuberosum tissues. However, evaluation of this relation is not explicitly shown in results. Results can only present plausible induction of bacterium gene expression by the plant tissues. Moreover, the article describes an in vitro set-up using injured plants. But, it is well-known that in vitro systems are not always allow the representation of the expected results in the presence of unbroken and real-size plants. It is a fact that infection processes have been demonstrated to be regulated or stimulated by host factors encountered in vivo. Therefore, this system also needs to be also applied in vivo.
Tn5-derived mini-transposons has been constructed to generate insertion mutants including in vivo fusions with reporter genes in Gram-negative bacteria a long time ago. Besides, high-throughput sequencing technologies are applied to study bacterial genome. Based on these, this method could and should be published and applied earlier. Authors, who are suggesting this method, have been working in the past in this field and as a result, they have the knowledge and the background to recommend such an approach. However, in order to see the reliability of the system, some example results from the follow-up studies, such as sequencing results would confirm it. Although they use an example of potato tissue which is known that interact with Dickeya species, the use of an already well-known up-regulated plant-induced gene and then quantification of its gene expression with fluorescence analysis will be a good suggestion. Eventually, the meaning of the cost-effective word is unprecedented, but they do not focus enough on the meaning of the words fast, reliable and efficient.
Some advantages of this method are mentioned by the authors. One of them is that recovery of bacterial cells after host infection is not required and this is what it makes it fast. But, no data and pieces of evidence are shown in their work for the recovery. Experiments that leads to this conclusion could be mentioned in the article. Another positive is that no expensive laboratory equipment and reagents are needed which make it cost-effective. Moreover, Tn5 mutants can be examined at the same time towards several factors unlike of microarrays and RNAseq. There is also no evidence data of the above report. Finally, the technique does not require any bioinformatics and statistical tools. Taking into consideration all the above, they concluded that it can be used in any microbiological laboratory.
The discovery and characterisation of plant-induced genes will provide insights for the demystification of the mechanism of bacterial pathogenicity at a molecular level. Thus, quick, smart and efficient methods will help to expand the studies around it. As in history, engineering of new technologies and the evolution of our understanding of bacterial pathogenicity at the level of the gene have a close relationship. The next phase in this studying would be an examination of this method in vivo. Currently, studies on bacterial pathogenicity using tissue culture as host defined some host-pathogen interactions and therefore will give the opportunity to solve the matrix of interactions sooner.