Mislabeled Microbes Cause Two Retractions

Two papers on plant immunity have been retracted, and questions remain about others with similar results. 

By Ed Yong | October 10, 2013

Rice plantsWIKPEDIA, IRRI IMAGESTwo samples of bacteria that were mislabeled several years ago have led to the retraction of two papers, including a highly-cited one published in Science.

In 2009, a team of scientists from the University of California, Davis, led by plant geneticist Pamela Ronald, identified a bacterial molecule that is recognized by the immune system of rice plants. It was the culmination of the lab’s longstanding quest to understand how this vital crop thwarts infections, and the paper has since been cited 131 times.

But when new lab members could not repeat the earlier results, the team discovered that one of the previous experiments had been done with mislabeled bacterial strains, while another had used an unreliable test. Ronald announced the problems to others in the field at conferences, and has now retracted the paper.

“There was never any question, even from the people who did the original work, that we do anything different than retract the paper if it was wrong,” said Ronald. “We didn’t want to mislead anyone else.”

The most common causes of scientific retractions are  misconduct or fraud, discovered through the actions of whistleblowers or watchful peers. It is rarer to see labs discover and own up to their own honest errors. “Too often, scientists, their institutions, and journals find ways to sweep painful reality under the rug, or make half-hearted attempts to correct the literature,”  says Ivan Oransky, a journalist who monitors scientific retractions through his blog Retraction Watch. “For a researcher to go out of her way to publicize her mistakes is unfortunately very unusual. Some scientists worry that retractions lead to a mistrust of science, but when handled appropriately the way Ronald’s have been, they only boost public confidence in research.”

In 1995, Ronald’s team showed that a receptor protein called XA21 allows rice plants to resist a devastating disease-causing bacterium called Xanthomonas oryzae. Many similar proteins were later discovered in humans, mice and other animals. Known as pattern recognition receptors (PRRs), their hallmark is the ability to recognise molecules found in a wide range of bacteria and trigger an immune reaction.

Nearly 15 years later, the team revealed that XA21 recognizes a bacterial protein called Ax21—the key to its lock. In a critical experiment, they showed that a mutant strain of X.oryzae, which lacked the gene for Ax21, can successfully infect rice leaves. Without this bacterial identity badge, XA21 could not mount an immune response. “The discovery made a lot of sense to us, and it seemed to fit everything that we and others had shown before,” said Ronald, whose results were published in 2009 in Science. “It was a very exciting time.”

The first hint of trouble came in June 2012. As is traditional for Ronald’s lab, a couple of new members tried to repeat the earlier experiments. But time and again, they failed. The new experiments repeatedly showed that rice plants could still resist X. oryzae strains that lacked Ax21.

“It’s tricky to work with rice, and they thought they were getting the experiments wrong,” said Ronald. But they eventually found that the 2 of the 12 original mutant strains had been mislabeled. Rather than Ax21, they were missing a different protein called RaxSt. “As far as we can figure out, it was just a mix-up,” said Ronald. “One team member gave it to another, and the one who did the experiment didn’t double-check. We should’ve caught it sooner.”

The revelation means that the molecule that XA21 recognizes is still a mystery. Ronald thinks that the mislabeled strains slipped past rice defenses because RaxSt modifies the unknown molecule, so that XA21 can recognize it. “We still don’t know what it is, and we’re trying to find it,” said Ronald. “It’s back to the drawing board.”

To make matters worse, the team found that another key result—that a synthetic version of Ax21 could trigger an immune response in rice—depended on an unreliable test, and could also not be consistently repeated.

Although Ronald feels that other aspects of the paper are still valid, she did not want the mistakes to waylay other labs while her team repeated the other experiments. She announced the problems at a plant immunity conference last April, and contacted Science to retract the paper. “I wanted to be sure that we got the word out early,” she said. The mislabeled strain was also used in experiments in a second paper, which was published in 2011 in PLOS ONE and was also retracted last month.

“Sorting out the situation was personally and professionally painful for all involved,” Ronald wrote in a new blog post. “Former lab members who had begun new positions as professors in Korea and Thailand were devastated to learn that [we] could not repeat their work. Junior scientists in the laboratory worried their careers would be tarnished. . . . It took persistence, courage and confidence to stick together as a team throughout this challenging year.”

The mistakes might never have been found at all if her lab did not commonly repeat old experiments, but Ronald is reticent to prescribe the same approach for other groups. “It’s just not realistic for most labs,” she told The Scientist. “Scientists move on, projects move on, there are funding issues. The bigger lesson is probably what we already know: you can’t rely on a single published report, or even more than that.”

Other scientists in the field have taken the retractions in good faith. “I feel absolutely confident that there was no intentional cheating,” said Markus Albert, a plant biologist from the University of Tubingen. Purifying and identifying the molecules that are recognized by PRRs “was, and still is, a difficult topic,” he said. “Otherwise, more of them would have been identified [by now].”

To complicate matters, at least three Chinese labs have successfully replicated Ronald’s Ax21 experiments. “I’ve talked to all the other laboratories, and they’re all confident in their results,” Ronald said. Wenxian Sun from China Agricultural University, who led one of the studies published online in Phytopathology, said that he has delayed formal publication until he can repeat the key results. The other teams did not respond to requests for comments.

“The fact that several labs have reported what might now be viewed as questionable data makes one pause,” said Sophien Kamoun, a plant biologist at the Sainsbury Laboratory. Along with the Chinese studies, he points to three other papers that are either “compromised or questionable” because they also involve Ax21: one from Ronald’s group, which found that XA21 gets cut and relocated after exposure to Ax21; one that lists Ronald as a collaborator, which found that Ax21 interacts with another immune receptor called FLS2; and a third from an independent lab, which found that some bacteria use Ax21 to communicate with each other—a similar result to Ronald’s now-retracted PLOS ONE paper. 

“I truly hope to see the authors of papers that are based on Ax21 come forward to clarify their work,” Kamoun said.

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Avatar of: Ellen Hunt

Ellen Hunt

Posts: 18

October 11, 2013

Very fishy. I'm sorry, but I have had too much experience with foriegn students to buy the "just an accident" explanation. I just don't believe that. I think Pamela was scammed, and she's a bit too trusting. Yes, Western students also fake and lie, but at far lower rates. 

As for the Chinese researchers who duplicated Pamela's work? That is way beyond fishy. That is a fish, dead and stinking, clamped around their necks. That is "caught red-handed". The possibility that their work is not fraudulent from top to bottom is zero. They faked it. Period. They should be chased from the profession. 

I would suggest deliberately publishing intentionally falsified papers, let them sit a few years, then pull the plug. Doing so would pull the frauds out of the woodwork. Anyone who bites and claims duplication of such papers should be criminally prosecuted for fraud. 

I am so sick of this gentlemanly, garden-club social rubbish when it comes to science fraudsters. We need some aggressive prosecutors out for blood. Enough with the scamming students, scamming PIs and the Gresham's law takeover of science by liars and cheats. 

Avatar of: mhstaufacar


Posts: 2

October 12, 2013

do they work for Monsanto?

Avatar of: mhstaufacar


Posts: 2

October 12, 2013

do they work for Monsanto?

Avatar of: GregGold


Posts: 3

Replied to a comment from Ellen Hunt made on October 11, 2013

October 14, 2013

Absolutely agree!

Avatar of: AGMMGA


Posts: 1

October 16, 2013


Rather than Ax21, they were missing a different protein called RaxSt.

"Ax21" and "RaxSt" would look very, very similar on the cap of an eppi or glycerol stock, especially after being thawed once or twice from the -20 / -80 freezer and thumbed a bit...

Smudge away the R, Ax == Ax and St ~= 21. And once you make the mistake and relabel your new tubes, you are done for...

Edit: that other groups could "reproduce" the finding is still very fishy, ofc...

Avatar of: Golden


Posts: 1

October 18, 2013

Dear everyone

As Dr Ed Yong earlier pointed out that three Chinese labs repeated the works of Ax21 in Science paper of Prof. Ronald, and listed our paper recently published in Journal of Proteomic Research (JPR). Here, as the corresponding author of JPR paper, I would like to clarify at this point.

We already contact with Prof. Ronald, telling her that we did not replicate the results of Ax21 in X. oryzae pv. oryzae (the bacterial pathogen of rice blight) performed by her Lab. We also did not perform any experiments described in Ronald’s science paper in X. oryzae pv. oryzicola (the bacterial pathogen of rice leaf streak). Our works in JPR only reported a gene (Xoc_0319) sharing sequence similarity to Ax21, is regulated by DSF signal and is involved in virulence in X. oryzae pv. oryzicola. Furthermore, we did not show experiments about Xoc_0319 activity on Xa21 rice, which is the main finding that has been retracted for Ronald’s Science paper. Thus, the website’s comment (https://www.the-scientist.com/?articles.view/articleNo/37843/title/Mislabeled-Microbes-Cause-Two-Retractions/) on our work is unjustified, which potentially mislead readers and ruin on our academic reputation as our reported works in X. oryzae pv. oryzicola are completely unrelated from Ronald's retraction work from Science and PLOS one. For details please see below:

My work is focused on the identification and functional characterization of extracellular proteins controlled by DSF (Diffusible-signal-factor) signal in X. oryzae pv. oryzicola. Recently, we used a 2-D proteomics to identify differentially expressed proteins between X. oryzae pv. oryzicola wild-type strain Rs105 and its DSF-deficient mutant, and obtained 33 DSF-controlled extracellular proteins. We then used a large-scale mutagenesis to identify which protein/gene of them was involved in virulence, and found 4 proteins/genes were virulence associated. Of them, the gene Xoc_0319 in X. oryzae pv. oryzicola was identified as a virulence factor on susceptible rice (Shanyou 63). Further sequence analysis showed that Xoc_0319 is the homologue of Ax21 (according to Ronald’s work in science, Ax21 from X. oryzae pv. oryzae can recognize the resistant gene XA21 in rice) in X. oryzae pv. oryzae, then based on this point, we defined Xoc_0319 as Ax21 in X. oryzae pv. oryzicola.

Subsequently, we investigated the contribution of Xoc_0319 in X. oryzae pv. oryzicola to virulence-associated functions. We also used QRT-PCR to confirm that Xoc_0319 in X. oryzae pv. oryzicola is controlled at transcriptional level by DSF signal. Recently, we used RNA-seq method to systematically identify the DSF regulons, and confirmed our previous finding (Xoc_0319 in X. oryzae pv. oryzicola is controlled at transcriptional level by DSF signal).

Overall, in our paper (Qian et al., 2013), we used a X. oryzae pv. oryzicola as a research target, and found that the Xoc_0319 is controlled by DSF signal, and is involved in virulence on susceptible rice (Shanyou 63). Based on our results in X. oryzae pv. oryzicola, and together with the reported cell-cell role of Ax21 in X. oryzae pv. oryzae (see retraction paper of PLos One), we assumed that Xoc_0319 might be a cell-cell signal protein in X. oryzae pv. oryzicola in the Discussion section.

My paper is provided as follows:

Guoliang Qian, Yijing Zhou, Yancun Zhao, Zhiwei Song, Suyan Wang, Jiaqin Fan , Baishi Hu , Vittorio Venturi, and Fengquan Liu. Proteomic Analysis Reveals Novel Extracellular Virulence-Associated Proteins and Functions Regulated by the Diffusible Signal Factor (DSF) in Xanthomonas oryzae pv. oryzicola. Journal of Proteomic Research, 2013, 12 (7): 3327-3341.

Avatar of: PCR


Posts: 1

Replied to a comment from Golden made on October 18, 2013

October 18, 2013

The papers from my laboratory were retracted solely because of errors we discovered in our own work. 

As Prof Liu points out, the experiments conducted in his laboratory were carried out completely independently of ours using differnet methods. There is no reason to question the quality of their work.

As soon as we noted problems with our strains. I contacted Professor Liu and Guoliang QIan to let them know. They were very helpful to us as we tried to sort through the issues in our lab and offered to share their expertise and strains.

Our new preliminary results supports their hypothesis that  rax genes  are regulated by DSF as they suggest in their paper.  We are also very interested in their observation that Ax21 is required for virulence in Xoc. My laboratory members will meet with Guoliang this week to discuss these experiments in detail and to consider additional experiments to elucidate Ax21 function.

Avatar of: jpizarro


Posts: 1

Replied to a comment from Ellen Hunt made on October 11, 2013

October 18, 2013

"Yes, Western students also fake and lie, but at far lower rates. "

I guess this resumes your post, science accuracy decreases dramatically as one moves east. Is this linked to the earth's magnetic field or some "faker" gene present at higher levels on the east?

PS - Did you read the three papers incriminated in the report? 

Avatar of: agular17


Posts: 5

December 7, 2013

Research science is pretty much on the way out anyway. The big bucks are in scam sciences, Global-Warming, all-whites-are-racist, male-gender-bashing and general trolling of mass databases for mass-media-agenda stuff. Oh, and ANYTHING that will get a Hollywoods attention.

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