| Xanthomonas oryzae pv. oryzicola (Xoc) is one of two pathovars in X. oryzae, causing serious bacterial leaf streak (Bacterial Leaf Streak, BLS) in rice in recent years in China. As other Gram-negative plant pathogenic bacteria, X. oryzae pv. oryzicola poseesses hrp genes to determines the ability to elicit hypersensitive response (HR) in nonhost plants and pathogenicity in host plants. The core hrp cluster of X. oryzae pv. oryzicola encodes a type-Ⅲsecretion system (T3SS), through which some of T3S effectors are secreted and translocated into plant cells to trigger HR in nonhost tobacco and to cause BLS symptoms in susceptible host rice. The 27 kb hrp cluster in X. oryzae pv. oryzicola consists of 10 hrp, 9 hrc (hrp-conserved) and 8 hpa (hrp-associated) genes (also named as hpa-hrp-hrc cluster) which expression is believed to be regulated by key regulatory gene hrpG and hrpX locating outside of the hrp cluster in the chromosome elswhere. Although the hpa-hrp-hrc genes of X. oryzae pv. oryzicola are highly conserved among species in Xanthomonas genus, their individual contribution to pathogenesis in rice is still not clear. In this study,29 single mutants correspondingly in the hpa-hrp-hrc genes and in two hrp regulatory genes hrpG and hrpX were generated, respectively, in X. oryzae pv. oryzicola wild-type strain RS105 as a recipient by using a suicide vector pKMS1. Based on the phenotypes observed on susceptible host rice (IR24) and nonhost tobacco (Xanthi) which were inoculated by these mutants, we found that all 9 hrc gene mutants lost Hrp phenotypes completely, that hpa gene mutants had no significant effect on the Hrp phenotype, but only hpaB and hpal gene mutant lost pathogenicity and reduced virulence on rice, respectively, and that in other ten hrp gene mutant, hrpF mutant retained HR in tobacco and reduced pathogenicity, the hrpE3 mutant retained equivalent pathogenicity in rice with the wild type, and the other hrp mutants including hrpG and hrpX lost Hrp phenotype. The above results provide further clues for understanding the precise roles of these hpa-hrp-hrc genes in X. oryzae pv. oryzicola-rice pathosystem. To investigate the regulation relationship between possible regulatory proteins and the hrp-hrc-hpa genes, we also knocked out other five global regulatory genes, zur, lrpX, colR, colS and trh, respectively, in X. oryzae pv. oryzicola, which have been reported to play roles in the expression of hrp genes in other Xanthomonas species. Pathogenicity assays in rice and HR induction in tobacco demonstrated that these five mutants reduced virulence in rice, but all kept the ability to trigger HR in tobacco.Though it is considered that HrpG and HrpX regulate the expression of hrp cluster in Xanthomonas, there are no debates that the expression of all the genes in the hrp-hrc-hpa cluster is not regulated by these two regulators in X. oryzae pv. oryzicola. In this work, transcriptional expression assays demonstrated that the expression of hrcC, hrpD5, hrpE and hpa3 genes were not dependent on HrpG and HrpX, the expression of hpa2 was positively regulated by HrpG, but not by HrpX while the expression of hrcT was positively regulated by HrpX but not by HrpG. Notably, in hrpD6 mutant, there was not transcriptional expression of hpa2, hpal, and hpaB, and the expression of hrcC and hrcT was down-regulated. The above suggests that the expression of hpa2, hpal, hrcC, hrcT and hpaB, not all, is regulated by HrpD6 which expression is positively regulated by HrpG and HrpX.To realize the influence of these seven regulatory proteins on the expression of the above genes, the putative promoters, phrcC for hrcC gene, phrcTl for hrpB operon, phrcT2 possible for hrcT gene within hrcN gene, phrpD51 for hrpD, phrpD52 possible for hrpD5 gene within hpaA gene and phpa3 for hpa3 operon were fused with a promoterless gusA gene which were introduced into X. oryzae pv. oryzicola RS105, and each transformant was incubated in rice suspension cells, a hrp-inducing medium XOM3 and the nutrient-rich medium NB for 16h, respectively, then (3-glucuronidase activity was measured. The results indicated that the transcription of hrcC, hrcT, hrpD5, hpa3 genes in X. oryzae pv. oryzicola is induced in the hrp-inducing conditions and repressed in nutrient-rich medium. On the other hand, the predicted promoters phrcT2 and phrpD52 were not hrp-inducing promoters.To preclude the possibility that these regulators are involved in regulation of hrpG, hrpX, hrcC, hrcT, hrpD5, hrpE and hpa3, the above reporter plasmids were introduced into the mutant strains△RhrpX,△RhrpG, ARzur,△RlrpX, ARcolR,△RcolS and△Rtrh and the wild-type RS105, respectively. The GUS activities of the resulting reporter strains were measured and showed that the hrpA operon expression was positively regulated by HrpG, Zur, ColR and Trh but not by HrpX, LrpX and ColS, that the hrpB operon was just positively regulated by HrpX, the hrpD operon was positively regulated by HrpX and HrpG, and that hrpG was just positively regulated by Trh, hrpX was positively regulated by HrpG, Zur and ColR but negatively by LrpX. However, phrcT2, phrpD52 and phpa3 were not regulated by all these seven regulatory proteins indicting that some unknown regulatory factor existed in X. oryzae pv. oryzicola to control the expression of hrcT, hrpD5, hrpE and hpa3 genes.To assess whether the expression of hrcC, hrpD5, hrcT, hrpE and hpa3 was down-or up-regulated or abolished in the mutants of hrpG, hrpX, zur, lrpX, colR/colS and trh genes, we then run Northern blot assay to detect the expression of the targets which indicated that hrpE was not regulated by HrpG, Trh, Zur and ColR, but positively regulated by HrpX and negatively regulated by LrpX. However, unfortunately, there were no hybridized signals appeared to display for hrcC, hrcT, hrpD5 and hpa3 transcripts, suggesting that the expression level of hrcC, hrcT, hrpD5 and hpa3 did not reach the amount for detection by Northern Blot.To analysis the transcript units characteristics of hrpD5 and hrpE, we first generated the knockout mutants of phrpD51 and phrpD52, respectively, and used RT-PCR to investigate the expression of individual genes from hrcQ to hrpE3 in ARhrpG, ARhrpX, ARhrpD51 and ARhrpD52 mutants, comparing those in the wild-type RS105. The results demonstrated that the expression of hrcR, hrcS, hrcQ, hpaA and hrpD6 genes was controlled by hrpD operon promoter phrpD51 as that in hrpG and hrpX mutants. Particularly, the hrpD5 expression was not affected as that in the wild-type RS105 when the promoters phrpD51 and phrpD52 and the hrpG and hrpX genes were mutated, implying that the hrpD5 expression is hrpG- and hrpX-independent and controlled by other unknown regulatory factor(s). Comparatively, the hrpE and hpaB genes could not express in phrpD51 promoter mutant, supporting that they are the members of hrpD operon. Then, we used RT-PCR to determine the transcriptional linkage in the region (hpaP through hrpE3) of the hrp gene cluster in RS105. The results indicated that the hrpD operon inclusive from hrcQ to hpaB, was transcribed as a single polycistronic mRNA. These results also proved that phrpD52 is just a promoter activity region but not the promoter for hrpD5 gene.In order to confirm whether or not HrpD6 has any regulatory relations with hpa2, hpal, hrcC, hrcT and hpaB, the fusion constructs of these genes promoters with gusA were introduced into the hrpD6 mutant and the wild-type RS105, respectively. The GUS activity of the resulting reporter stains showed that HrpD6 regulated the expression of hpa2, hpal and hpaB and partially controlled the transcript of hrcC and hrcT genes, while HrpD6 had no obvious influence on the hrpG or hrpX expression in X. oryzae pv. oryzicola.To detect the expression of hpa2 and hpal affected by HrpD6 on protein level, we used X. oryzae pv. oryzicola strain RS105, hrpD6 mutant△RhrpD6, a T3SS deletion mutant△RhrcV and a hpaB deletion mutant ARhpaB to analyze Hpa2 and Hpal secretion in vitro. The results showed that Hpa2 and AvrXa27 were not detectable in the culture supernatants of hpaB and hrpD6 deletion mutants, respectively, and Hpal was only secreted in the culture supernatant of HpaB deletion mutant, and that HrpF secretion was observed both in hpaB and hrpD6 deletion mutants, suggesting that the mutation in hrpD6, resulting in no transcriptional expression of hpa2 and hpal, leads to no detected proteins of Hpa2 and Hpal secreted. Since HrpD6 positively regulates the hpaB expression, it is reasonable that HpaB-dependent T3S effectors AvrXa27 and Hpa2 are not secreted in hrpD6 mutant, while the secretion HpaB-independent translocator, HrpF, is not affected by HrpD6. Best to our knowledge, it is the first report that HrpD6 is a regulatory factor for the hrp-hrc-hpa cluster in Xanthomonas species.In X. oryzae pv. oryzicola RS105, hpa2 with an imperfect PIP-box in the promoter region, locates at the left border of the core hrp cluster and produces a lytic transglycosylase. hrpF locates at the right border of the core hrp cluster and the gene product is putative thought as a translocator through which T3SS effectors are translocated into plant cells. Whether Hpa2 interacts with HrpF or not to form a complex to control T3SS effector translocation for pathogenicity of X. oryzae pv. oryzicola in rice is still unknown. In this study, we knocked out hpa2 and hrpF genes in X. oryzae pv. oryzicola and found that the double mutation in hpa2 and hrpF led to the complete loss of pathogenicity in rice, but kept the maintenance of HR induction in tobacco, while the single mutagenesis in either hpa2 or hrpF resulted in reduced virulence in rice,hrp-inducing assays revealed that the expression of hpa2 was regulated by hrp regulator HrpG, but not completely by HrpX. Furthermore, transient expression with green fluorescence protein (GFP) in onion cells displayed that Hpa2 could be located into plant cell membrane. In addition, protein-protein interaction and secretion assay demonstrated that Hpa2 was secreted through T3SS machinery and interacted with the traslocator HrpF, suggesting that Hpa2 and HrpF may form a complex to control effectors, like AvrXa27, to be translocated into plant cells.In X. oryzae pv. oryzicola, the expression of hrp genes, generally within promoter regions containing a PIP-box (plant-inducible promoter, TTCGC-N15-TTCGC), are regulated by HrpX. To study the regulatory mechanism that hpal regulated by HrpX by in vivo strategy, the promoter region of hpal was fused with the reporter gene gfp (green fluorescence protein), giving a recombination plasmid carrying phpal:gfp. The plasmid was then introduced into X. oryzae pv. oryzicola wild-type strain RS 105 and hrpX mutant and the reporter strains were incubated with rice cells for 16h. Uner fluorescence microscope, the fluorescent bacterial cells was observed in the wild-type RS105, while there were not fluorescent bacterial cells displayed in hrpX mutant which carried the plasmid (phpal:gfp), indicating that HrpX regulates the expression of gfp under the PIP-promoter of hpal gene. Correspondingly, the RT-PCR also demonstrated that hpal was not expressed in hrpX mutant, verifying that hpal was regulated by HrpX.Harpins are heat-stable, glycine-rich, typeⅢsecreted proteins produced by plant pathogenic bacteria, which cause HR when infiltrated into the intercellular space of tobacco leaves. So far the hpal gene product is the only known Harpin in X. oryzae pv. oryzicola. However, the biochemical mechanisms that Harpins elicit plant programmed cell death remains unclear. To determine what the regions of X. oryzae pv. oryzicola Hpal are critical for HR induction in nonhost plants, we generated truncated-Hpal derivatives and key amino acid mutation in Hpal. The mutants of Hpal and the wild-type Hpal were used to complement the double mutant△Rhpal△hrpF which lost HR on tobacco. The results indicated that the N-terminal a-helix of Hpal were essential for HR induction in tobacco, while the C-terminalα-helix of Hpal had no such the ability. Specially, the mutation in the site of the 47th (C) and the 53th (L) amino acids led to the loss of HR induction in tobacco. Furthermore, transient expression with green fluorescence protein (GFP) in onion epidermal cells displayed that Hpal could locate in plant cell membrane which was determined by the N-terminal a-helix of Hpal. Based on the specific recognition between Xa10 and AvrXa10 in X. oryzae pv. oryzae-rice pathosystem which was used as a reporter, the first 60 amino acids of the N-terminal of Hpal was fused with AvrXa10 reporter lacking secretion signal and the construct was introduce into X. oryzae pv. oryzae PXO99A, which could trigger resistance response on IRBB10 containing Xa10. Consequently, Hpal could be secreted from bacteria via T3S machinery which was consistent with the Western blot result. All these findings would provide more favorable clues for non-host resistance.Since the PIP-box sequence is highly conserved in Xanthomonas species, it is believed that the sequence can be used as an effective screening marker to obtain HrpX regulons from the genome database of X. oryzae pv. oryzicola. For this, we here established a platform to identify HrpX regulongs from X. oryzae pv oryzicola. First, five candidate genes(hrpB1, avrBs2, ecpA, kgtP and Z1234) were taken as the targets, either containing PIP-box (perfect or imperfect) or not. The promoters of these five genes were fused with reporter gene gfp in the vector pURF034. The combinated plasmids were introduced into the wild-type RS105 and hrpX mutant of X. oryzae pv. oryzicola, respectively, and the reporter strains were incubated in rice suspension cells for 16 h. Under fluorescent microscope, the promoter containing perfect or imperfect PIP-boxes promoted gfp gene to express in the wild-type strain, but not in hrpX mutant. This was also confirmed by RT-PCR. The results above suggest that the genes with PIP-box promoter are HrpX regulons. These novel PIP-gfp strategy may facilitate the screening for unknown HrpX regulons in X. oryzae pv. oryzicola.Regarding that Hpal of X. oryzae pv. oryzicola activates HR on nonhost plant tobacco and induces some beneficial attributions, such as anti-disease, anti-pest and enhancing plant growth, to plants when it is applied as a biochemical agent, it seems that Hpal can be transgenic into plants. Since Hpal has not bacterial-inhibiting activity, our lab developed a fusion protein of Hpal with cecropin A (CA) and Meltin (ME) active domains and named it as Hcml. To realize Hcml expressed in rice, in this study, the Hcml and Hpal (as a control) were constructed under CaMV 35S promoter, pathogen-induced expressed promoters hsr203J and PR]a, giving recombined constructs pBIhcml, pBIhshcml and pBIPRhcml respectively, and then were introduced into rice cultivar (Wujinl5) by Agrobacterium tumefacient EHA105. PCR and Southern blot confirmed that hcml and hpal had integrated into the chromosome of receptor rice and Northern blot demonstrated that hcml and hpal was expressed in transgenic rice lines. Additionally, Northern blot also revealed that defense-related genes PRla and NPR1 could be activated in the transgenic rice, suggesting that the defense genes were activated in hpal and hcml transgenic rice, respectively. More importantly, some of the obtained transgenic rice lines were highly resistant to rice blast (Magnaporthe grisea) and rice sheath blight(Rhizoctonia solani). |
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