Tobacco resistance gene expression levels in response to the infection of Phytophthora nicotianae

One of the main obstacles often encountered in efforts to increase productivity and quality of tobacco (Nicotiana tabacum) yields is the attack of black shank disease by the oomycetes Phytophthora nicotianae. So far, what has been done mainly by tobacco cultivators is the observation of resistance through calculating the rate of death or disease of plants due to pathogen attack, so research is needed to determine the resistance profile of local varieties of tobacco to black shank disease in molecular stage to speed up the screening process of tobacco varieties. This study aim to determine the gene expression profile related to resistance in tobacco varieties Beinhart 1000, Hick Broadleaf, and two local varieties, namely Dark 302 and Dark 314, in response to black shank disease. The research consisted of preparing tobacco and P. nicotianae inoculum, artificial inoculation, followed by semi-quantitative analysis of gene expression related to tobacco resistance, PR1, PR4, PLP2, and PUB24. Tobacco resistant variety, Beinhart 1000, expressed a relatively 285% higher PR1 significantly than the control. The length of time of infection showed that PR1, PR4, PLP2, and PUB24 genes were expressed temporally, and the dynamics of expression of each test gene affected the development of pathological conditions.


Introduction
"Lanas", also known as the black shank, caused by the oomycetes Phytophthora nicotianae, is a major disease of tobacco worldwide.Phytophthora nicotianae is a soil pathogen from the Stramenopiles-Alveolata-Rhizaria subgroup [1].This pathogen can spread rapidly under conditions of 23-28C temperature and high soil moisture [2].This disease is detrimental to farmers because it can attack tobacco plants at all ages, both as seedlings and adults, especially in the root, stem, and leaf organs [2][3][4].Symptoms of infection that arise are wilting plants, the base of the stem being rotten and appearing brownish, and the appearance of insulation in the pith when splitting [5].Research that has been done before showed the influence of local Indonesian tobacco varieties with lanas disease resistance, namely 4 out of 10 varieties (Dark A, Dark B, Jepon Pote, and Marakot) were determined to be more resistant to P. nicotianae attack [6].Four tobacco varieties will be studied in this research, namely Beinhart-1000, Hick Broadleaf, Dark 302, and Dark 314.Beinhart-1000 is one of the tobacco varieties resistant to P. nicotianae infection, while Hick Broadleaf is an example of a susceptible variety [7].The specific resistance of Dark 302 and Dark 314 tobacco varieties to P. nicotianae has not yet been studied.So far, what has been done by tobacco farmers in the field is the observation of resistance by calculating the level of death or illness of plants due to pathogen attacks.Therefore, it is necessary to research to determine molecularly the gene expression profile associated with tobacco resistance to malignant disease to accelerate the screening of tobacco varieties.The selected tobacco plant resistancerelated genes are Pathogenesis related 1 (PR1), Pathogenesis related 4 PR4 [4], while susceptibilityrelated genes are Patatin-like protein 2 (PLP2) and Plant U-box 24 (PUB24) [8].The purpose of this study is to determine whether there are differences in the level of gene expression in tobacco varieties Beinhart-1000, Hick Broadleaf, Dark 302, and Dark 314 in response to infection with P. nicotianae, as well as the effect of the length of time of infection with P. nicotianae on gene expression.The hypothesis formulated for this study is that there are differences in gene expression related to resistance to Phytophthora nicotianae infection in tobacco plants of Beinhart-1000, Hick Broadleaf, Dark 302, and Dark 314 varieties.

Plants materials
Observations and preliminary research were carried out from August to December 2021, and the main research continued from January to July 2022.The research was conducted in the Laboratory of Biotechnology Microorganism, Laboratory of Purification and Molecular Biology, and greenhouse of the Laboratory of Plant Biotechnology, Faculty of Biotechnology, University of Surabaya.The independent variables studied in this study were tobacco plant varieties, namely Beinhart-1000, Hick Broadleaf, Dark 302, and Dark 314.The dependent variable observed was the relative expression of PR1, PR4, PLP2, and PUB24 in the test tobacco, with the L25 gene as the housekeeping gene.The control variables in this study were the amount of inoculant soil given as an infectious agent, the age of tobacco of each variety, the sampling time point, and the growth conditions of tobacco in the greenhouse.

Tobacco preparation
Tobacco seedlings were seeded by sprinkling 0.07 g of tobacco seeds evenly onto the wet soil surface.After 14 days after sowing (das), the seedlings were transferred to the nursery tray using tweezers.At 60 das, the tobacco seedlings were transferred to polybags containing 10 liters of growing media.The growing media used consisted of a mixture of compost fertilizer and media mix in a ratio of 5:1.The media mix contained river soil, cocopeat, and husk charcoal in a ratio of 4:3:2.Tobacco fertilization was done by applying 25 mL of KNO fertilizer solution3 with a concentration of 1.5 g/L to the soil of each polybag.Tobacco seedlings planted in the polybags were maintained until 144 das before the next treatment was finally carried out.In this study, 48 tobacco plants were used, of which 24 were included in the treatment group while the rest were controlled.

Isolation of Phytophthora nicotianae culture from infected tobacco
Candidate cultures of Phytophthora nicotianae were isolated from the stems of tobacco plants experiencing symptoms of malignant disease in the field.The culture isolation method refers to the method conducted by [9].The tobacco stems from being taken were first washed with running water, split, and surface sterilized in 0.5% sodium hypochlorite solution for 30 s, then rinsed using sterile distilled water.The inside of the dried stem was then placed in Corn Meal Agar (CMA) medium aseptically to be incubated for 1-2 days at room temperature (±25 o C).Subculturing was done three times to purify the candidate culture from contaminant microorganisms.
Macrogen carried out molecular identification of isolate species through the Internal Transcribed Spacer (ITS) Region Sequencing program.The ITS region of ribosomal DNA (rDNA) was amplified using a pair of common primers, namely ITS4 and ITS5.The primer sequences used were ITS5 (5'-TCC GTA GGT GAA CCT GCG G-3') and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3').The ITS1-5.8S-ITS2 sequences obtained were then analyzed using the BLAST tool on the NCBI web server.

Preparation of Phytophthora nicotianae starter inoculum
Rolled oat grains needed to grow cultures were sterilized by autoclaving at 121 o C for 15 min.After that, 5 agar culture plugs were aseptically placed into a small jar containing 25 g of oat grains, and 10 mL of sterile distilled water was added.Five small culture jars of oat grains were incubated in the dark for 5 days at room temperature.Then the 5 small jars of oat grains culture were aseptically transferred to a larger jar containing 250 g of oat grains and 100 mL of sterile distilled water.Incubation was carried out for 20 days.A total of 365 g of oat grains culture was mixed with 1.926 g of soil taken from around the roots of tobacco infested with malaria in the field, forming an inoculum concentration of 18.95% w/w to be inoculated into the treated tobacco.

Artificial inoculation and tobacco leaf sampling
Artificial inoculation of tobacco was done by sprinkling and mixing the surface soil of tobacco growing media with 95 g of inoculum.Artificial inoculation was carried out in the afternoon in the rainy season when the air humidity was high (78.25±5)%,while the air temperature was low (26.2±1.3)o C, thus favoring disease infection by Phytophthora nicotianae.Young non-necrotic tobacco leaves from each variety, both control and treatment groups, were taken on days 0, 1, 2, 3, and 9 after artificial inoculation (dai = days after inoculation) of P. nicotianae inoculum.The leaves were picked, put into aluminum foil bags, and then liquid nitrogen was added to freeze the leaves.

RNA extraction and cDNA synthesis
According to the manual instructions, RNA extraction was performed using an RNA extraction kit (Favorgen, FavorPrepTM Plant Total RNA Mini Kit).Approximately 100 mg of tobacco leaf powder was placed in a 1.5 mL microtube for total RNA extraction.Complementary DNA (cDNA) chains were prepared from tobacco RNA extracted at the previous stage using reverse transcription.In this study, the SMOBIO Reverse Transcription Kit RP1400 was used according to the manual instructions.Tobacco cDNA solution is stored in a freezer at -20 o C until the time to be used.

Gene expression analysis
PR1, PR4, PLP2, and PUB24 expression were analyzed using the PCR method followed by electrophoresis.The composition of the mixture used was 1 µL of cDNA template that had been diluted twice, 0.5 µL of forward primer (10 µM), 0.5 µL of reverse primer ( 10 The cycle used for PR1 amplification was 28 cycles, while PR4, PLP2, and PUB24 were 36 cycles.Gene expression analysis results using PCR and electrophoresis methods produce data in black-and-white photos of amplicons from the gel observed under UV light.Calculating relative gene expression begins with analyzing the thickness or intensity of the amplicons using ImageJ software that has been calibrated to produce data in the form of peak areas.The thickness of the amplicons of the test genes, namely PR1, PR4, PUB24, and PLP2, was compared with the housekeeping gene, L25, to obtain the relative expression value of the gene.

Analysis of sequencing results
Sequence correction of sequencing results was carried out with the help of SeqMan Pro DNAStar software.Sequencing results from both primers, namely ITS4 and ITS5 primers, were entered, then combined through the "Assemble" menu and observed the combined results (contigs) that were successfully obtained.Contig sequences were identified using the BLAST Nucleotide (BLASTn) NCBI web server.A Neighbor-Joining phylogenetic tree showing the relationship between the isolated culture ITS region sequencing results and the ITS regions of Lasiodiplodia pseutheobromae and Phytophthora nicotianae was created using Molecular Evolutionary Genetics Analysis (MEGA) 11 software.

Statistical analysis
Statistical data analysis was carried out with the help of IBM SPSS Statistics 26 software.The data normality test used the Kolmogorov-Smirnov method, followed by the data homogeneity test, then the test for differences in gene relative expression was carried out using the Mann-Whitney method because the data was found to be not normally distributed, although homogeneous.Data is called significantly different when it has a P value <0.05.

Black shank disease infection in tobacco
Root tobacco sections from the treatment groups, Beinhart-1000, Hick Broadleaf, Dark 302, and Dark 314 varieties, were inoculated with soil from the area around the roots of malignant tobacco in the field.Disease incidence was observed from the day of inoculation (0 dai), until 50 days later (50 dai).Based on observations, Hick Broadleaf tobacco experienced sudden death at 3 dai (experienced by 3 out of 6 plants), followed by dying at 9 dai.(Fig. 1A), and ended in death at 20 dai.Symptoms of stem rot and stem base rot were also experienced by Dark 302 (all plants) and Dark 314 (3 out of 6 plants) tobacco varieties, observed at 20 dai."Lanas" symptoms were observed on all Dark 314 tobacco varieties at 30 hsi and died at 50 dai.The lower stems of Hick Broadleaf, Dark 302, and Dark 314 tobacco varieties were split to observe the formation of lesion borders (Fig. 1B).In contrast, until the observation at 50 dai, Beinhart-1000 tobacco appeared healthy and without disease symptoms, indicating its resistance to infection.

Expression Analysis of PR1, PR4, PLP2, and PUB24
A temporal comparison of the relative expression of PR1, PR4, PLP2, and PUB24 can be observed in Figure 2. A significant increase in PLP2 expression against the control group occurred in Dark 302 tobacco at 2 dai, which was 124% higher than the control.A significant increase in PUB24 expression against the control group occurred in Hick Broadleaf tobacco at 3 dai, 83% greater than the control.In tobacco variety Beinhart-1000, there was a significant increase in PR1 expression at 1 dai, reaching 285% higher than the control, where this phenomenon was not observed in the other 3 varieties.Interestingly, at 3 dai, the day when the first symptoms of senescence appeared in the susceptible variety, Hick Broadleaf, little PR1 expression was observed, even the lowest among the other 3 varieties, which was 64% of the control.The significant increase in PR1 at 1 hsi is a response to Beinhart-1000's resistance to malignant disease infection, where it is known that PR1 is a downstream marker of the defense response controlled by the salicylic acid signaling pathway [10].A significant increase in PR4 expression against the control group occurred in Dark 314 tobacco varieties at 1 dai, which increased by 161% compared to the control.In field practice, Dark 314 is a medium-resistant variety, so this significant increase in PR4 data indicates a different defense strategy between Beinhart-1000 and Dark 314.The increase in PR4 is related to the role of PR4 as a downstream marker of the defense response mediated by the jasmonic acid and ethylene signaling pathways [10].

Isolate culture sequencing results
Analysis of the sequencing results performed by Macrogen shows the sequence similarity between the isolated culture and Lasiodiplodia pseutheobromae, which has a sequence similarity identity of 99%, evalue 0.0, and coverage 96%.The results of sequence improvement and phylogenetic tree analysis also show the same conclusion, namely, the isolated culture has a close relationship with the species Lasiodiplodia pseutheobromae, so it is identified as Lasiodiplodia pseutheobromae.So far, there have been no reports of L. pseutheobromae infection in tobacco.It is suspected that the dominant pathogenic agent in the soil mixture and infested oats that could attack the test tobacco was P. nicotianae spores, while L. pseutheobromae, an endophytic organism that can grow in plant tissues without causing disease symptoms, was isolated when taking samples from diseased tobacco stems.

Persistence of Phytophthora nicotianae in soil and environmental factors for successful P. nicotianae infection
Phytophthora nicotianae inoculated on the test tobacco in this study need to invade host cells to obtain nutrients and fulfill its life cycle, so it has strategies and attack weapons that support it in infecting susceptible tobacco varieties effectively.On the other hand, in response to biotic stress from phytopathogenic microorganisms, tobacco develops a self-defense system that aims to limit the movement and resist the growth of pathogens, namely in the form of pathogen-associated molecular patterns-triggered immunity (PAM-TI/PTI), and effector-triggered immunity (ETI).The interaction dynamics of the tobacco immune response against pathogen attack involves the expression of various phytohormones and proteins that support tobacco resistance and susceptibility.In this study, it was shown that differences in the expression profiles of several resistance genes (PR1, PR4) and susceptibility (PLP2, PUB24) related genes among tobacco varieties temporally early in the infection period might affect the success of tobacco against P. nicotianae infection.
Soil from tobacco growing sites infected with "Lanas" can be used as an infectious agent due to the presence of spores of Phytophthora spp., which can survive in the soil for a long time [11], 6 years for chlamydospores and 4 years for oospores, even after the tobacco tissue has decomposed.Hyphae and sporangia, and later zoospores, can develop naturally from oospores and chlamydospores when the soil is moist.When suitable host plants are present, either sporangia, hyphae or zoospores can infect the host, thus restarting the disease cycle.The inoculum of P. nicotianae oospores and chlamydospores that persist in the soil for a long time often become the main and primary inoculum that causes malaria in the field [8].It seems that the strategy of using spore survival is successfully applied in this study; soil inoculation can cause symptoms of malignant disease in tobacco because spores survive.Appropriate environmental factors are also required for successful pathogen infection, where P. nicotianae infection has been successfully carried out on 30-40 days old tobacco in an environment with a temperature of 22-26 o C and relative humidity of 65-70%.[1].The results of thermometer and hygrometer measurements in the afternoon during the sampling period showed that the greenhouse where the test tobacco was infected had an average temperature of 26.2 o C and a relative humidity of 78.25%, thus supporting the infection of P. nicotianae into the roots of the test tobacco.
The successful invasion of P. nicotianae is characterized by its ability to invade the active cambium in the stem and colonize nearby transport tissues [12].Lack of nutrients and water caused the test tobacco variety Hick Broadleaf leaves to wilt at 3 dai.At 9 dai, it was observed that the continuous inhibition of nutrient distribution caused stem tissue to die, characterized by dryness at the base of the stem and discoloration from green to brown, as well as severe leaf chlorosis (Fig. 1A).Further invasion of P. nicotianae results in the death of all tobacco plant tissues, the formation of insulation in the dead stem vessel network, as well as root rot.This progression of malignant disease infection was also observed in Dark 314 and Dark 302 varieties but at a longer period than Hick Broadleaf.This indicates the role of varietal differences in the defense response to P. nicotianae infection.

PR1 role and expression in P. nicotianae-treated-tobacco plant defense
PR1 plays an important role in tobacco defense against P. nicotianae because it can act as a sterol binder important in the growth and development of P. nicotianae as a sterol auxotroph organism.PR1 can bind sterols in the plasma membrane of P. nicotianae through the C-terminal domain of the cysteine-rich secretory protein (CAP), causing the death of the pathogen [13].PR1 is a small size of 4 kDa protein.
In addition to being secreted outside the cell to interact with P. nicotianae, high concentrations of PR1 directly were also found to accumulate in the cell vacuole as a more effective advanced defense that is launched when the pathogen successfully penetrates the plant cell wall [14].Overexpression of PR1 protein has been widely reported to increase plant resistance to P. nicotianae by binding sterols [15], but the molecular mechanism to obtain this resistance is not yet known.
In this study, PR1 gene expression in the Beinhart-1000 variety increased significantly at 1 dai, whereas this event was not observed in the other 4 varieties, either at 1 hsi or all sampling points.The achievement of peak PR1 gene expression in the first 24 hours after inoculation is in line with the results of Ali et al. (2018) on Altenaria brassicae infection in Brassica juncea and Li-Xia et al. (2012) on Plasmopara viticola infection in Vitis vinifera [16,17].While Bao et al. (2019) observed that PR1 expression in resistant tobacco variety TKF2022 inoculated with P. nicotianae continued to increase from 0 hours after inoculation (hai), 12 hai, 24 hai (1 dai), and reached peak expression at 72 hai [4].High PR1 expression generally indicates the Systemic Acquired Resistance (SAR) response by Beinhart-1000 tobacco varieties to suppress the growth of the biotrophic phase of P. nicotianae, in addition to sterol binding activity by PR1 [18].Systemic Acquired Resistance can protect uninfected tissues, so the distribution of SA to leaves conveys signals for expressing defense-related genes, such as PR1.It seems that this increase in PR1 at the beginning of P. nicotianae infection has successfully suppressed the growth of the pathogen so that the formation of P. nicotianae colonies is not maximized; the infection of tobacco fails.Another supporting research proved that the growth of Phytophthora brassicae was inhibited by 90% by treatment with PR1 protein [19].The opposite condition occurs in Dark 302, Dark 314, and susceptible varieties, namely Hick Broadleaf, which express little PR1, making it easy to be resisted by P. nicotianae colonies [20].Other results are supported by the research of Bao et al. (2019) where PR1 gene expression by susceptible tobacco variety TKF4321 was observed to increase dramatically at 96 hai or 4 dai [4].This is thought to be due to differences in genotypes between tobacco varieties affecting the ability of tobacco to respond to SA signals into PR1 transcription.

PR4 role and expression in P. nicotianae-treated-tobacco plant defense
The N-terminal hevein-like (CB-HEL) domain of PR4 can bind to chitin on the cell wall of thereby supporting host resistance to pathogens such as fungi [21].The cell wall of the oomycetes P. nicotianae is not composed of chitin, but cellulose (β-1,4-glucan), β-1,3 and β-1,6-glucans [22].However, previous studies have shown that overexpression of PR4 protein confers resistance to P. nicotianae [23].This is supported by another activity of PR4 besides chitinase, which is the degradation of pathogenic RNA (ribonuclease; RNAse) by the C-terminal hevein-like (CD-HEL) domain of PR4.The presence of amino acid residues His11 and His110 on the active side of the CD-HEL domain of Arabidopsis PR4 provides ribonuclease activity that strengthens host resistance to fungal pathogens, namely through the mechanism of cutting phosphodiester bonds according to the acid-base reaction of enzymes such as RNAse A [21,24].
In this study, a significant increase in PR4 expression occurred against the control by Dark 314 tobacco varieties at 1 dai, and was observed to be maintained higher than the control until 3 dai.This suggests that the activity of the tobacco defense response through the JA and ET pathways affects the time course of malignant disease development, which counteracts the development of P. nicotianae so that symptoms only appear at 20 dai.Susceptible varieties, namely Hick Broadleaf, do not express much PR1 and PR4, two types of PR protein groups that can support plant resistance, so pathogen growth is poorly controlled.Increased expression of PR4 in the resistant medium tobacco, Dark 302, was observed from 9 his.This result is similar to the results of Bao et al. (2019) that after being inoculated with P. nicotianae, the resistant tobacco variety, TKF2002, expressed more PR4 than the susceptible variety, and maintained its high expression until 3 dai [4].Another study by Bai et al. (2013) showed that PR4 transcription in Malus domestica (MdPR4) increased by 8.58 times from the control in response to B. dothidea inoculation at 30 dai [25].
Many researchers have described the antagonistic relationship between SA and JA pathway plant defense signaling, as well as the synergism of JA and ET.JA signaling can reduce SA accumulation by activating the transcription factor NAC, thus inhibiting the expression of isochorismate synthase 1 (ICS1), which is a SA biosynthesis gene [26].This can explain the event that occurred in this study, when PR1 was highly expressed at 1 hsi by Beinhart-1000 tobacco, but decreased dramatically the next day, replaced by highly expressed PR4 at 2 dai, representing the inhibition of SA accumulation.Although JA/ET defense pathways, including PR4 expression, generally play a role against necrotrophic organisms, previous studies have found its expression responds to SA induction, also against hemibiotrophic organisms such as P. nicotianae early in its life cycle because it is expressed higher than the control starting 6 h after inoculation [4,25].Increased expression of PR1 and PR4 in tobacco infected with P. nicotianae has also been known to be in line with increased production of H O22 which acts as a detoxification response to increased levels of reactive oxygen species (ROS) in the PTI response [4].

PLP2 role and expression in P. nicotianae-treated-tobacco plant defense
Patatin-like protein 2 (PLP2) has a catalytic dyad consisting of serine and aspartic acid amino acid residues on its active side to perform its function as a lipid acyl hydrolase (LAH).In response to pathogens, NtPLP2 can act as a modulator gene that promotes cell death, and is also involved in oxylipin metabolism starting from the hydrolysis of chloroplast membrane galactolipids and phospholipids.Silencing of PLP2 gene expression is known to increase Arabidopsis resistance to the necrotrophic organism B. cinerea and the hemibiotrophic P. syringae while wild type Arabidopsis experiences more severe disease symptoms [27].In contrast, hydrolysis of phospholipids or galactolipids can produce fatty acids as precursors of the defense reaction jasmonic acid, thereby increasing the resistance of Arabidopsis to cucumber mosaic virus (CMV) infection.The process of cell death in plant defense mechanisms is tightly controlled and balanced to maximize pathogen growth restriction through hypersensitive reactions, but also inducing defenses against pathogens.PLP2 activity can increase in the presence of necrosis stimuli so that more cell death occurs, which is when the balance shifts in favor of the hypersensitive reaction (HR).
High expression of PLP2 by Dark 302 and Hick Broadleaf tobacco at 2 hsi may play a role in tobacco susceptibility to P. nicotianae infection; in line with the previous report [8,27].When associated with the possibility ki nan hydrolase results to be a precursor in the biosynthesis of jasmonic acid, the event can be observed at 9 hsi where the expression pattern of PLP2 coincides with PR4 which is a marker gene of the JA signaling pathway.Tobacco variety Dark 302 expressed the highest amount of PLP2 in relative terms, and PR4 was also observed; while Hick Broadleaf expressed the least.This indicates that high JA activity is supported by an adequate supply of its precursors from hydrolysis by PLP2 protein.Phospholipids or galactolipids can be hydrolyzed and produce products in the form of fatty acids (polyunsaturated fatty acid; PUFA), then catalyzed by lipoxygenase (LOX) enzyme activity to produce FA-hydroperoxide, then metabolized into JA [28].

PUB4 role and expression in P. nicotianae-treated-tobacco plant defense
The plant u-box E3 ubiquitin ligase protein (PUB24) plays an important role in the ubiquitin-proteosome pathway, which can determine the specificity of protein targets to be degraded through its substraterecognizing domain.In the context of plant defense, ubiquitin ligases can play a role in regulating the concentration of defense-related proteins so that they only accumulate when the plant is under stress, then eliminate them when they are no longer needed, for example in regulating the elimination of the phosphorylated WRKY70 transcription factor (WRKY70-P) when there is no pathogen attack [29].However, several studies have shown that the ligase function of E3 PUB24 can also play a negative role in the plant defense system against pathogens, including P. nicotianae.
The increased expression of E3 PUB24 in the test tobacco indicates the influence of susceptibilityrelated genes during the development of disease infection.The results of this study are in line with the results of Meng et al. (2021) as well as Trujillo et al. (2008), which concluded that the relative expression of E3 PUB24 in tobacco variety XHJ increased from 6 hai, reached peak expression at 12 hai, and was kept high until 60 hai played a role in increasing plant susceptibility by suppressing the PTI response [8,30], Mutations in PUB24 (pub24) from Arabidopsis inoculated with the H. parasitica caused an extension of the duration and accumulation of ROS due to increased expression of the gene encoding the ROS production enzyme, RbohD, as well as an increase in marker genes for PTI, namely OX1, WRKY29 [30].On the other hand, oomycetes can secrete effectors to suppress the PTI response of plants, such as Avr3a protein from P. infestans.This synergism of pathogenic effectors and negative effects of E3 PUB24 explains the relationship between highly expressed E3 PUB24 in test tobacco and tobacco susceptibility.The time at which the host defense response is activated plays a role in determining host susceptibility and resistance because host susceptibility can occur not only due to the host's inability to express resistance-associated proteins, but also too late to inhibit the growth of pathogens have already colonized.Differences in gene expression dynamics between tobacco varieties in this study have provided a molecular basis for the resistance of the resistant variety Beinhart-1000 and the susceptible variety Hick Broadleaf to P. nicotianae, as well as a comparison with the local varieties Dark 302 and Dark 314.In this study, 6 test tobacco varieties of Beinhart-1000 were observed to be healthy until the 50th day after inoculation (50 dai).Beinhart-1000 tobacco actively expressed resistance-related genes, namely PR1 starting at 1 hsi and PR4 starting at 2 dai.
IOP Publishing doi:10.1088/1755-1315/1255/1/0120499 4.6.Genetic distance analysis and disease symptoms in P. nicotianae-treated-tobacco Genetic distance analysis showed that the average kinship score between the land isolate culture and P. nicotianae was 0.7344 while with L. pseutheobromae was 0.0051.The smaller score means that the kinship between the isolate culture and L. pseutheobromae is closer, that is, there are fewer sequence differences, in this case there are at least 5 different bases in each 1000 bases analyzed.However, the disease symptoms caused in the test tobacco in this study did not match the disease symptoms caused by Lasiodiplodia pseutheobromae, namely dieback on the trunk and branches, splitting of the bark and wedge-shaped rot on woody trees [31], as well as gummosis, thus supporting the statement that L. pseutheobromae may be an endophytic fungus that was picked up during culture isolation.

Conclusion
Throughout infection, gene expression dynamics continue to occur and affect tobacco defense in addition to P. nicotianae resistance.PR1, PR4, PLP2, and PUB24 are just 4 of the many genes involved in tobacco defense.The resistant tobacco variety, Beinhart-1000, expressed PR1 at a relative level of 285%, significantly higher than the control.The mechanism of tobacco resistance and susceptibility given from the results of this study is a theoretical conjecture, so it needs to be further investigated to the proteomic level.The length of time of infection shows that genes are temporally expressed and the dynamics of expression of each test gene affect the development of pathological conditions.To further this research, focus on proteomic analysis and identification of additional resistance genes while exploring gene expression dynamics, environmental factors, and epigenetics.Develop molecular markers for resistance breeding to efficiently develop new tobacco cultivars with improved resistance to black shank disease.

Figure 1 .
Figure 1.The appearance of malignant disease symptoms; Hick Broadleaf tobacco dying, experiencing malignant disease symptoms of chlorosis, stem rot, and decay at 9 dai (A); Necrotic schist and hyphae at the base of a longitudinally sectioned tobacco stem (B).

Figure 2 .
Figure 2. Relative expression graph of PR1, PR4, PLP2, and PUB24 genes from Beinhart-1000, Hick Broadleaf, Dark 302, and Dark 314 tobacco varieties.Error bars show the standard deviation of 3 replicates.Expression levels are normalized to the housekeeping gene (L25) and relative expression of the control group.Notes: *P < 0.05: significantly different from control.A significant increase in PR4 expression against the control group occurred in Dark 314 tobacco varieties at 1 dai, which increased by 161% compared to the control.In field practice, Dark 314 is a medium-resistant variety, so this significant increase in PR4 data indicates a different defense strategy between Beinhart-1000 and Dark 314.The increase in PR4 is related to the role of PR4 as a downstream marker of the defense response mediated by the jasmonic acid and ethylene signaling pathways[10].